Recombinant light chains of botulinum neurotoxins and light chain fusion proteins for use in research and clinical therapy

ABSTRACT

Botulinum neurotoxins, the most potent of all toxins, induce lethal neuromuscular paralysis by inhibiting exocytosis at the neuromuscular junction. The light chains (LC) of these dichain neurotoxins are a new class of zinc-endopeptidases that specifically cleave the synaptosomal proteins, SNAP-25, VAMP, or syntaxin at discrete sites. The present invention relates to the construction, expression, purification, and use of synthetic or recombinant botulinum neutoroxin genes. For example, a synthetic gene for the LC of the botulinum neurotoxin serotype A (BoNT/A) was constructed and overexpressed in  Escherichia coli.  The gene product was purified from inclusion bodies. The methods of the invention can provide 1.1 g of the LC per liter of culture. The LC product was stable in solution at 4° C. for at least 6 months. This rBoNT/A LC was proteolytically active, specifically cleaving the Glu-Arg bond in a 17-residue synthetic peptide of SNAP-25, the reported cleavage site of BoNT/A. Its calculated catalytic efficiency k cat /K m  was higher than that reported for the native BoNT/A dichain. Treating the rBoNT/A LC with mercuric compounds completely abolished its activity, most probably by modifying the cysteine-164 residue located in the vicinity of the active site. About 70% activity of the LC was restored by adding Zn 2+  to a Zn 2+ -free, apo-LC preparation. The LC was nontoxic to mice and failed to elicit neutralizing epitope(s) when the animals were vaccinated with this protein. In addition, injecting rBoNT/A LC into sea urchin eggs inhibited exocytosis-dependent plasma membrane resealing.

[0001] This application is a continuation-in-part of U.S. patentapplication Ser. No. 09/910,186 filed Jul. 20, 2001, which is acontinuation of U.S. patent application Ser. No. 09/611,419 filed Jul.6, 2000, which is a continuation of U.S. patent application Ser. No.08/123,975, filed Sep. 21, 1993, wherein said application 09/611,419 isbased on U.S. Provisional Applications Nos. 60/133,866, 60/133,868,60/133,869, 60/133,865, 60/133,873, and 60/133,867, all filed May 12,1999, all of which are incorporated herein by reference in theirentirety. The instant application is also based on U.S. ProvisionalApplication No. 60/246,774, filed on Nov. 6, 2000, and U.S. ProvisionalApplication No. 60/311,966, which are incorporated herein in theirentirety by reference.

FIELD OF THE INVENTION

[0002] This invention is directed to construction, expression, andpurification of synthetic DNA molecules encoding polypeptides comprisingbotulinum neurotoxin (BoNT) light chains. The invention is also directedto methods of vaccination against botulism using the expressed peptides.

BACKGROUND OF THE INVENTION

[0003] The sporulating, obligate anaerobic, gram-positive bacillusClostridium produces eight forms of antigenically distinct exotoxins.Tetanus neurotoxin (TeNT) is produced by Clostridium tetani whileClostridium botulinum produces seven different neurotoxins which aredifferentiated serologically by specific neutralization. The botulinumneurotoxins (BoNT) have been designated as serotypes A, B, C₁, D, E, F,and G. Botulinum neurotoxins (BoNT) are the most toxic substances knownand are the causative agents of the disease botulism. BoNT exert theiraction by inhibiting the release of the neurotransmitter acetylcholineat the neuromuscular junction (Habermann, E., et al., (1986),“Clostridial Neurotoxins: Handling and Action at the Cellular andMolecular Level,” Cur. Top. Microbiol. Immunol., 129:93-179; Schiavo,G., et al., (1 992a), “Tetanus and Botulinum-B Neurotoxins BlockNeurotransmitter Release by Proteolytic Cleavage of Synaptobrevin,”Nature, 359:832-835; Simpson, L. L., (1986), “Molecular Pharmacology ofBotulinum Toxin and Tetanus Toxin,” Annu. Rev. Pharmacol. Toxicol.,26:427-453) which leads to a state of flaccid paralysis. Indeed, only afew molecules of toxin are required to abolish the action of a nervecell. Polyclonal antibodies derived for a specific neurotoxin canneutralize the toxic effects of that toxin but will not cross-neutralizeanother toxin serotype. Thus, to protect against all seven toxins, oneneeds seven vaccines.

[0004] Human botulism poisoning is generally caused by type A, B, E orrarely, by type F toxin. Type A and B are highly poisonous proteinswhich resist digestion by the enzymes of the gastrointestinal tract.Foodborne botulism poisoning is caused by the toxins present incontaminated food, but wound and infant botulism are caused by in vivogrowth in closed wounds and the gastrointestinal tract respectively. Thetoxins primarily act by inhibiting the neurotransmitter acetylcholine atthe neuromuscular junction, causing paralysis. Another means forbotulism poisoning to occur is the deliberate introduction of thetoxin(s) into the environment as might occur in biological warfare or aterrorist attack. When the cause of botulism is produced by toxin ratherthan by in vivo infection the onset of neurologic symptoms is usuallyabrupt and occurs within 18 to 36 hours after ingestion. The most commonimmediate cause of death is respiratory failure due to diaphragmaticparalysis. Home canned foods are the most common sources of toxins. Themost frequently implicated toxin is toxin A, which is responsible formore than 50% of morbidity resulting from botulinum toxin.

[0005] Botulinum and tetanus neurotoxins are a new class ofzinc-endopeptidases that act selectively at discrete sites on threesynaptosomal proteins of the neuroexocytotic apparatus. See Montecuccoand Schiavo, 1995, and Schiavo, 1995, for review. These neurotoxins arethe most potent of all the known toxins. The botulinum neurotoxins(BoNT), designed A-G, produced by seven immunologically distinct strainsof Clostridium botulinum cause death by flaccid muscle paralysis at theneuromuscular junction. Extreme toxicity of these toxins and theirlability in purified preparations have limited any detailedcharacterizations.

[0006] These neurotoxins are expressed as 150-kDa single polypeptides(termed dichains) containing a disulfide bond between the 50-kDaN-terminal light chain (LC) and the 100-kDa C-terminal heavy chain (HC).A post-translational cryptic cleavage generates the two chains connectedby a disulfide bond. The LC contains the toxic, zinc-endopeptidasecatalytic domain. The 100-kDa HC may be further proteolyzed into a50-kDa N-terminal membrane-spanning domain (H_(n)) and a 50-kDaC-terminal receptor-binding domain (H_(c)).

[0007] With three functional domains, the mechanism of action of theseneurotoxins is multiphasic: (1) The H_(c) domain plays a role in bindingthe toxins to specific receptors located exclusively on the peripheralcholinergic nerve endings (Black and Dolly, 1986). (2) The H_(n) domainis believed to participate in a receptor-mediated endocytotic poreformation in an acidic environment, allowing translocation of thecatalytic LC into the cytosol. Reducing the disulfide bond connectingthe LC with the H upon exposure to the cytosol or within the acidicendosome (Montal et al., 1992) releases the catalytic LC into thecytosol. (3) The LC then cleaves at specific sites of one of the threedifferent soluable NSF attachment protein receptor (SNARE) proteins,synaptobrevin, syntaxin, or synaptosomal associated protein of 25 kDa(SNAP-25) (Blasi et al, 1993; Schiavo et al., 1993, 1994; Shone et al,1993; Foran et al, 1996). These proteins are essential for synapticvesicle fusion in exocytosis. Their proteolysis inhibits exocytosis andblocks acetylcholine secretion, leading ultimately to muscularparalysis. The LC itself is nontoxic because it cannot translocatethrough the cholinergic nerve ending into the cytosol. However, indigitonin-permeabilized chromaffin cells, the LC inhibits exocytosis(Bittner et al., 1989), and direct microinjection of the LC into thecytosol results in blockage of membrane exocytosis (Bittner et al.,1989; Bi et al., 1995).

[0008] The LC of all known clostridial neurotoxins contain the sequenceHExxH that is characteristic of zinc-endoproteinases (Thompson et al.,1990). The essential role of zinc on the structure and catalysis of theneurotoxins is established (Fu et al., 1998). A unique feature of theneurotoxins' protease activity is their substrate requirement. Shortpeptides encompassing only the cleavage sites are not hydrolyzed (Foranet al., 1994; Shone and Roberts, 1994). A specific secondary and/ortertiary structure of the substrate is most probably recognized(Washbourne et al., 1997; Lebeda and Olson, 1994; Rossetto et al., 1994)rather than a primary structure alone, as is the case with most otherproteases. Most importantly, their identified natural substrates areproteins involved in the fundamental process of exocytosis (Blasi etal., 1993; Schiavo et al., 1993, 1994; Shone et al., 1993; Foran et al.,1996). Light chain also is the target of an intensive effort to designdrugs, inhibitors, and vaccines. A detailed understanding of itsstructure and function is thus very important.

[0009] The present invention describes the construction andoverexpression of a synthetic gene for the nontoxic LC of BoNT/A in E.coli. The high level of expression obtained enabled purification of gramquantities of LC from 1 L of culture as well as extensivecharacterization. The preparation of the rBoNT/A LC was highly soluble,stable at 4° C. for at least 6 months, and had the expected enzymaticand functional properties. For the first time, a cysteine residue wastentatively identified in the vicinity of the active site which, whenmodified by mercuric compounds, led to complete loss of enzymaticactivity.

[0010] The BoNTs and their LCs are targets of vaccine development, drugdesign, and mechanism studies because of their potential role inbiological warfare, wide therapeutic applications, and potential tofacilitate elucidation of the mechanism of membrane exocytosis. In spiteof such immense importance, studies of the LC have been limited by itsavailability. Commercially available LC is prepared by separating itfrom the dichain toxins under denaturing conditions. These preparationstherefore retain some contaminating toxicity of the dichain, have lowsolubility, and often begin to proteolytically degrade and start losingactivity within hours of storage in solution.

[0011] The LC of serotype A has been separated and purified from thefull-length toxin by QAE-Sephadex chromatography from 2 M urea; however,the preparation suffers from low solubility (Shone and Tranter, 1995).The LC of serotype C was similarly obtained at a level of <5 mg/10 Lculture of C. botulinum (Syuto and Kubo, 1981). These preparationsalmost invariably contain contaminating full-length toxins, and thecommercially available preparations precipitate from solution or undergoproteolytic degradation upon hours of storage in solution. More recentlythe LC of tetanus neurotoxin (Li et al., 1994) and of BoNT/A (Zhou etal., 1995) were expressed in E. coli as maltose-binding proteins andpurified in 0.5 mg quantities from 1-L cultures (Zhou et al., 1995).However, the poor expression of the cloned products, probably due torare codon usage in clostridial DNA (Makoff et al., 1989, Winkler andWood, 1988), remained a major hurdle in obtaining adequate amount of theprotein for structural and functional studies.

[0012] Most of the clostridial strains contain specific endogenousproteases which activate the toxins at a protease-sensitive loop locatedapproximately one third of the way into the molecule from theamino-terminal end. Upon reduction and fractionation(electrophoretically or chromatographically), the two chains can beseparated; one chain has a Mr of ˜100 kDa and is referred to as theheavy chain while the other has a Mr ˜50 kDa and is termed the lightchain.

[0013] The mechanism of nerve intoxication is accomplished through theinterplay of three key events, each of which is performed by a separateportion of the neurotoxin protein. First, the carboxy half of the heavychain (fragment C or H_(c) is required for receptor-specific binding tocholinergic nerve cells (Black, J. D., et al., (1986), “Interaction of¹²⁵I-botulinum Neurotoxins with Nerve Terminals. I. UltrastructuralAutoradiographic Localization and Quantitation of Distinct MembraneAcceptors for Types A and B on Motor Nerves,” J. Cell Biol.,103:521-534; Nishiki, T.-I., et al., (1994), “Identification of ProteinReceptor for Clostridium botulinum Type B Neurotoxin in Rat BrainSynaptosomes,” J. Biol. Chem., 269:10498-10503; Shone, C. C., et al.,(1985), “Inactivation of Clostridium botulinum Type A Neurotoxin byTrypsin and Purification of Two Tryptic Fragments. Proteolytic ActionNear the COOH-terminus of the Heavy Subunit Destroys Toxin-BindingActivity, Eur. J. Biochem., 151:75-82). Evidence suggests thatpolysialogangliosides (van Heyningen, W. E., (1968), “Tetanus,” Sci.Am., 218:69-77) could act as receptors for the toxins but the datasupporting a specific receptor remains equivocal (Middlebrook, J. L.,(1989), “Cell Surface Receptors for Protein Toxins,” BotulinumNeurotoxins and Tetanus Toxin, (Simpson, L. L., Ed.) pp. 95-119,Academic Press, New York). After binding, the toxin is internalized intoan endosome through receptor-mediated endocyctosis (Shone, C. C., etal., (1987), “A 50-kDa Fragment from the NH₂-terminus of the HeavySubunit of Clostridium botulinum Type A Neurotoxin Forms Channels inLipid Vesicles, Euro. J Biochem., 167:175-180).

[0014] The amino terminal half of the heavy chain is believed toparticipate in the translocation mechanism of the light chain across theendosomal membrane (Simpson, 1986; Poulain, B., et al., (1991),“Heterologous Combinations of Heavy and Light Chains from BotulinumNeurotoxin A and Tetanus Toxin Inhibit Neurotransmitter Release inAplysia,” J Biol. Chem., 266:9580-9585; Montal, M. S., et al., (1992),“Identification of an Ion Channel-Forming Motif in the Primary Structureof Tetanus and Botulinum Neurotoxins,” FEBS, 313:12-18). The low pHenvironment of the endosome may trigger a conformational change in thetranslocation domain, thus forming a channel for the light chain.

[0015] The final event of intoxication involves enzymatic activity ofthe light chain, a zinc-dependent endoprotease (Schiavo, 1992a; Schiavo,G., et al., (1992b), “Tetanus Toxin is a Zinc Protein and its Inhibitionof Neurotransmitter Release and Protease Activity Depend on Zinc,” EMBOJ, 11:3577-3583), on key synaptic vesicle proteins (Schiavo, 1992a;Oguma, K., et al., (1995), “Structure and Function of Clostridiumbotulinum Toxins,” Microbiol. Immunol., 39:161-168; Schiavo, G., et al.,(1993), “Identification of the Nerve Terminal Targets of BotulinumNeurotoxin Serotypes A, D, and E,” J Biol. Chem., 268:23784-23787;Shone, C. C., et al., (1993), “Proteolytic Cleavage of SyntheticFragments of Vesicle-Associated Membrane Protein, Isoform-2 by BotulinumType B Neurotoxin,” Eur. J Biochem., 217:965-971) necessary forneurotransmitter release. The light chains of BoNT serotypes A, C₁, andE cleave SNAP-25 (synaptosomal-associated protein of M25,000), serotypesB, D, F, and G cleave vessicle-associated membrane protein(VAMP)/synaptobrevin (synaptic vesicle-associated membrane protein); andserotype C₁ cleaves syntaxin. Inactivation of SNAP-25, VAMP, or syntaxinby BoNT leads to an inability of the nerve cells to releaseacetylcholine resulting in neuromuscular paralysis and possible death,if the condition remains untreated.

[0016] The majority of research related to botulinum toxin has focusedon the development of vaccines. Currently, a pentavalent toxoid vaccineagainst serotypes A through E (Anderson, J. H., et al., (1981),“Clinical Evaluation of Botulinum Toxoids,” Biomedical Aspects ofBotulism, (Lewis, G. E., Ed.), pp. 233-246, Academic Press, New York;Ellis, R. J., (1982), “Immunobiologic Agents and Drugs Available fromthe Centers for Disease Control. Descriptions, Recommendations, AdverseReactions and Scrologic Response,” 3rd ed., Centers for Disease Control.Atlanta, Ga.; Fiock, M. A., et al., (1963), “Studies of Immunities toToxins of Clostridium botulinum. IX. Immunologic Response of Man toPurified Pentavalent ABCDE Botulinum Toxoid,” J. Immunol., 90:697-702;Siegel, L. S., (1988), “Human Immune Response to Botulinum Pentavalent(ABCDE) Toxoid Determined by a Neutralization Test and by anEnzyme-Linked Immunosorbent Assay,” J. Clin. Microbiol., 26:2351-2356),available under Investigational New Drug (IND) status, is used toimmunize specific populations of at-risk individuals, i.e., scientistsand health care providers who handle BoNT and military personnel who maybe subjected to weaponized forms of the toxin. Though serotypes A, B,and E are most associated with botulism outbreaks in humans, type F hasalso been diagnosed (Midura, T. F., et al., (1972), “Clostridiumbotulinum Type F: Isolation from Venison Jerky,” Appl Microbiol.,24:165-167; Green, J., et al., (1983), “Human Botulism (Type F)—A RareType,” Am. J. Med., 75:893-895; Sonnabend, W. F., et al., (1987),“Intestinal Toxicoinfection by Clostridium botulinum Type F in an Adult.Case Associated with Guillian-Barre Syndrome,” Lancet, 1:357-361;Hatheway, C. L., (1976), “Toxoid of Clostridium botulinum Type F:Purification and Immunogenicity Studies,” Appl. Environ. Microbiol.,31:234-242). A separate monovalent toxoid vaccine against BoNTF isavailable under IND status. Hatheway demonstrated that the BoNTF toxoidcould protect guinea pigs against a homologous challenge (Wadsworth, J.D. F., et al., (1990), “Botulinum Type F Neurotoxin,” Biochem. J,268:123-128).

[0017] New-generation, recombinant vaccines have also been developed byUSAMRIID (e.g. Dertzbaugh M T, Sep. 11, 2001, U.S. Pat. No. 6,287,566;U.S. Appln. Ser. No. 09/910,186 filed Jul. 20, 2001; and U.S. Appln.Ser. No. 09/611,419 filed Jul. 6, 2000) and commercial sources (e.g.Ophidian Pharmaceuticals, Inc. Williams J A, Jul. 6, 1999, U.S. Pat. No.5,919,665; using clones supplied by USAMRIID).

[0018] Most vaccine studies have focused on the botulinum toxin heavychain, leaving the light chain largely ignored. In 1995, Zhou et al.discovered that a single mutation in the light chain of botulinumneurotoxin serotype A abolished its neurotoxicity and its ability tocleave SNAP-25, one of the natural substrates, when reconstituted withthe heavy chain. See Zhou, L. et al., (1995), “Expression andPurification of Botulinum Neurotoxin A: A Single Mutation Abolishes itsCleavage of SNAP-25 and Neurotoxicity after Reconstitution with theHeavy Chain,” Biochem., 34:15175-15181.) This raised the possibilitythat the mutated light chain might have various research or therapeuticuses. Further research produced a recombinant light chain (Li, L. andSingh, B. R., (1999), “High-Level Expression, Purification, andCharacterization of Recombinant Type A Botulinum Neurotoxin LightChain,” Protein Expression and Purification, 17:339-344) and a constructcomprising the minimum essential light chain domain (Kadkhodayan, S., etal., (2000), “Cloning, Expression, and One-Step Purification of theMinimal Essential Domain of the Light Chain of Botulinum Neurotoxin TypeA,” Protein Expression and Purification, 19:125-130).

[0019] Recombinant production methods alleviate many of the problemsassociated with the toxoid, such as the need for a dedicatedmanufacturing facility. Presently, many cGMP facilities are in existenceand available that could manufacture a recombinant product. There wouldbe no need to culture large quantities of a hazardous toxin-producingbacterium. Production yields from a genetically engineered product areexpected to be high. Recombinant products would be purer, lessreactogenic, and more fully characterized. Thus, the cost of arecombinant product would be expected to be much lower than a toxoidbecause there would be no expenditures required to support a dedicatedfacility, and the higher production yields would reduce the cost oftherapeutic and research products.

[0020] However, recombinant methods as described in the publicationsabove do not yield optimal results because botulinum codons are nottranslated well in other organisms commonly used for production, such asE. coli or yeast. Furthermore, no easily translatable, recombinant formof the non-neurotoxic, mutated light chain presently exists. Recombinantforms of both functional and non-neurotoxic botulinum neurotoxin thatmay be translated efficiently in either E. coil or yeast are needed forresearch and therapeutic purposes.

[0021] Commercially available BoNT LC is prepared by separation from thedi-chain toxins. These preparations, therefore, retain somecontaminating toxicity, have low solubility, and undergo proteolyticdegradation within hours and days of storage in solution. Many clinicaldisorders are presently being treated with a botulinum neurotoxincomplex that is isolated from the bacterium, Clostridium botulinum.There is no data to demonstrate that the binding proteins play any rolein the therapeutic effects of the drug. The binding proteins, however,probably contribute to the immunological response in those patients thatbecome non-responsive to drug treatment. Recombinant products could bemanufactured under conditions that are more amenable to productcharacterization. Chimeras of the drug product could also be produced bydomain switching. Chimeras could potentially increase the number ofpotential useful drug products.

[0022] Recently, the BoNT LC of serotype A has been expressed as amaltose-binding protein and purified in 0.5 mg quantities from 1 literculture (Zhou et a., 1995). The poor expression of the native gene wasprobably due to the high A+T composition found in the clostridial DNA.

SUMMARY OF THE INVENTION

[0023] The present invention relates to the design and construction ofsynthetic DNA molecules that encode one of the seven light chains ofClostridium botulinum neurotoxin and are capable of being expressed inheterologous prokaryotic or eukaryotic hosts. The invention is based, inpart, on modifying the wild-type BoNT sequence according to the codonusage normally found in genes that are highly expressed in the hostorganism. By selecting codons rich in G+C content, the synthetic DNAmolecules may further be designed to lower the high A+T rich basecomposition found in clostridial genes.

[0024] The invention further relates to methods of expressing andpurifying recombinant BoNT light chains. According to the invention,BoNT LC may be expressed in a heterologous host system by itself or as afusion to another protein or carrier. For example, the BoNT LC may befused to a synthetic or wild-type BoNT heavy chain or a fragmentthereof. BoNT LC of the invention may or may not have catalytic activityas a zinc protease. In some embodiments of the invention, catalyticallyinactive BoNT LC is fused to a BoNT heavy chain forming a mutantholotoxin. Non-enzymatic, non-toxic mutant holotoxins are capable ofbeing internalized into nerve cells. In addition, mutant holotoxins maybe used as transporters to carry other molecules into colinergic nervecells.

[0025] The invention further provides methods and compositions foreliciting an immune response to BoNT LC and BoNT HN. The inventionprovides preparations of BoNT LC and BoNT HN that are capable ofelicting protective immunity in a mammal.

BRIEF DESCRIPTION OF THE DRAWINGS

[0026]FIG. 1. Nucleotide sequence of rBoNT/A LC and the correspondingamino acid sequence. The codon in italics (i.e., encoding thepenultimate val residue) and at the 5′ end of the gene was introduced tocreate and maintain the Nco I restrictions enzyme site. Codon in italics(i.e., encoding LVPRGS) at the 3′ end of the gene encode a thrombinprotease cleavage site for removing the His tag after purification.

[0027]FIG. 2. SDS-PAGE followed by Coomassie stain (A) and Western blot(B) of crude and purified BoNT/A LC expressed in E. coli containing thesynthetic gene for BoNT/A LC in a multicopy plasmid pET24. Totalcellular protein (T), soluble supernatant (S), insoluble pellet (P), andpurified inclusion bodies (I) were prepared as described in Section 2.Lane 1 shows Novex wide-range molecular-mass markers (0.8-3.0 μg/band).The sarkosyl solubilized inclusion bodies of the LC had the sameelectrophoretic behavior as (I). About 20 μg of protein was applied perlane. Western blot used affinity-purified rabbit polyclonal antibodiesagainst a 16-residue N-terminal sequence of the BoNT/A LC as the primaryantibody and a peroxidase-coupled goat anti-rabbit IgG (H+L) as thesecondary antibody. Bands were visualized by a chromogenic substrate.

[0028]FIG. 3. UV-visible absorption spectrum of the rBoNT/A LC.

[0029]FIG. 4. Long-term stability at 4° C. (A) and thermal stability (B)of the rBoNT/A LC. (A) Aliquots of the LC from one single preparationwere assayed at the indicated times; (B) 50 μl aliquots of the LC inbuffer G containing 1 mM DTT and 501 tM ZnCl₂ were taken in Eppendorftubes and heated for 5 min at the indicated temperatures. After coolingon ice for 60 min, the supernatants were assayed for proteolyticactivity.

[0030]FIG. 5. Proteolysis of the synthetic peptide substrate by therBoNT/A LC. The peptide (1.1 mM) was incubated for 5 min (A) or 200 min(B) with the rBoNT/A LC. The reaction products were analyzed byreverse-phase HPLC. The first three peaks represent the solvent front(<4 min) and reduced DTT (5.2 min) in the reaction mixture. Sequence ofthe substrate and the sequences of the products are shown in panels Aand B, respectively. The numbers above the sequences represent the LCresidue numbers corresponding to the sequence of SNAP-25. The productpeaks (not labeled in Panel A) were identified by sequence determinationby MS-MS.

[0031]FIG. 6. Effect of pH on the endopeptidase activity of the rBoNT/ALC. Activities were measured at various pH of 0.1 M buffers: MES (-◯-),HEPES (--), and tris-HCl (-Δ-) containing 0.9 mM substrate peptideMaximum activity (100%) was 334 nmol/min/mg LC.

[0032]FIG. 7. Inhibition of endopeptidase activity of the rBoNT/A LC byexcess Zn²⁺ and protection from inhibition by DTT. The LC was assayed inSO mM HEPES, pH 7.4, containing 0.9 mM substrate peptide in the absence(-◯-) and presence of 5 mM DTT (--) or 5 mM mercaptoethanol (-Δ-)containing the indicated concentrations of ZnC₂. One hundred percentactivity (290 nmol/min/mg LC) represents the activity obtained in theabsence of any added thiol or Zn²⁺.

[0033]FIG. 8. Determination of K_(m) and V_(max) from thedouble-reciprocal (Lineweaver-Burke) plot of initial rates ofproteolysis versus substrate concentration by the rBoNT/A LC. Thereaction mixtures (0.03 ml) contained 0.25 mM ZnCl₂, 0.5 mM DTT, 50 mMHEPES, pH 7.4, and 0.016 mg rBoNT/A LC. The K_(m) and V_(max) werecalculated as 0.9 mM and 1500 nmol/min/mg, respectively.

[0034]FIG. 9. Location of the three Cys residues in the BoNT/A LC.Molecular surface of the LC portion of the BoNT/A dichain based on itsthree-dimensional structure (Lacy and Stevens, 1999) is shown. The threeCys residues (yellow), active-site His and asp residues (red), the Zn²⁺atom (blue) at the active site, and the ‘pit’ leading to the active siteare highlighted. The side chain of Cys-164 lines the surface and formspart of the wall of the ‘pit’ leading to the active site. The ‘pit’ actsas an access route of the substrate.

[0035]FIG. 10. Time course of proteolysis of BoNT/A LC as followed bySDS-PAGE (A) and Western blot (B). Aliquots of 25 ml of the LC (0.2mg/ml) were incubated at 4° C. At intervals (see below), 25 μl of2×SDS-load buffer was added to an aliquot and boiled. Two SDS gels wererun in parallel. One gel was stained by Coomassie (A) and the proteinsfrom the other were transferred to a nitrocellulose membrane for Westernblot (B). Lane 1 in panel A shows Novex Mark-12 molecular weight markersand lane 1 in panel B shows the Novex prestained SeeBlue molecularweight markers. In both panels A and B, lanes 2-7 show 0, 2, 4, 14, 21,and 28 days of incubation, respectively, of LC. Identity of the proteinbands between panels A and B is arbitrary, and the same nomenclature isused throughout the paper.

[0036]FIG. 11. Enhancement of the proteolysis of BoNT/A LC by ZnCl₂ asfollowed by SDS-PAGE (A) and Western blot (B). All conditions are sameas in FIG. 1, except that 0.25 mM ZnCl₂ was added to the incubationmixture of the LC.

[0037]FIG. 12. Protection of BoNT/A LC from proteolysis by the metalchelator TPEN (A) and the competitive peptide inhibitor CRATKML (B),followed as a time course by SDS-PAGE. (A) the LC (0.2 mg/ml) wasincubated in small aliquots with 10 mM EDTA (lanes 2-5) or with 5 mMTPEN (lanes 7-10). Lanes 2 and 7, 3 and 8, 4 and 9 and 5 and 10 show 6,14, 21, and 28 days of incubation, respectively, (B) The LC wasincubated with 1 mM peptide inhibitor containing 5 mM DTT (lanes 2-5) orwithout the peptide inhibitor (lanes 10-7) at 4° C. DTT, which does nothave an effect on proteolysis, was added to maintain the peptide inmonomer form. Lanes 2 and 10, 3 and 9, 4 and 8, and 5 and 7 show 6, 14,21 and 28 days of incubation, respectively. In both panels A and B, lane1 represents LC alone at day 0, and lane 6 has molecular weight markers(labels on left). The protein band IIIA (see FIG. 1) was faint in thisexperiment and was not captured in the photographic reproduction;therefore its location in the original gel is shown by arrows in thefigure. Note that (a) presence (lanes 2-5, A) and absence (lanes 10-7,B) of EDTA had little effect on proteolysis of IA to IB and finally toIIIA, (b) TPEN (lanes 7-10, A) significantly reduced the rate ofconversion of IA to IB and prevented formation of IIIA during the courseof the experiment, and (c) the peptide inhibitor (lanes 2-5, B)drastically reduced the proteolysis of IA to IB and prevented theformation of IIIA.

[0038]FIG. 13. Scheme I. Steps in the self-proteolysis of BoNT/A LC inthe absence of added zinc. Arrows show the sites of proteolysis.Full-length LC is denoted by IA. The fragments IB, IIIB, and IVCcorrespond to the fragment designations in FIG. 1. The primary event isthe C-terminal truncation to form IB followed by cleavage between Y286and G287 producing IIIA and IVC. The fragment IIIA in turn is furtherproteolyzed between Y251 and Y252 to generate IIIB. Lengths of thefragments (e.g., IV-K448) are based on mass determined by MALDI-MS andN-terminal amino acid sequence shown in Table 5. The C-terminal peptideE424-K448, although shown here as a single peptide for convenience, isin fact a mixture of several peptides (see Tables 4 and 5).

[0039]FIG. 14. Scheme II. Steps in the self-proteolysis of BoNT/A LC inthe presence of added zinc. Arrows show the sites of proteolysis. Thefragments IIIB, IVA, and IVB correspond to the fragment designations inFIG. 2. Unlike the steps shown in Scheme I, IA may bypass the C-terminaltruncation and initial formation of IIIA but undergo proteolysis betweenY251 and Y252 in directly forming IIIB. The fragment IVA is furthercleaved into IVB. Although a C-terminal cleavage of IVB into IVC ispossible, it was not observed here (see FIG. 11) this species in thepresence of added zinc. See FIG. 11 and Scheme I for other explanations.

[0040]FIG. 15. SDS-PAGE of (A) LCA, (B) LCA+Belt, and (C) LCA+Xloc,expressed at 18° C., 30° C. and 37° C. Odd numbered lanes (1, 3, 5 and7) are the soluble fractions and even number lanes (2, 4, 6 and 8) arethe insoluble fractions. Lanes 7 and 8 are control cells with theplasmid lacking the insert. Arrows show the expressed product at 18° C.(soluble) and 37° C. (insoluble).

[0041]FIG. 16 (FIG. 2, Manuscript). HPLC elution profiles from HS columnof LcA (A, B), LcA+Belt (C, D), LcA+Hn (E, F), and LcB (G,H) and from aSource S column of LcA (I, J).

[0042]FIG. 17. SDS-PAGE (A) and Western blots of purified LcA constructsusing rabbit peptide sera against LcA (B), LcA+Belt (C) and LcA+Hn (D).Lanes from all figures are identical. Lane 1, Novex See Blue prestainedmolecular weight markers; Lane 2, purified BoNt-A; Lane 3, LcA-HIS; Lane4, LcA-phosphate buffer; Lane 5, LcA-NaAcetate buffer; Lane 6, LcA+Belt;Lane 7, LcA+Hn, nicked; Lane 8, LcA+Hn, un-nicked; Lane 9, negativecontrol pET24a construct, no insert; Lane 10, LcB.

[0043]FIG. 18. Mass spectrum for cleaved BoNT/A Lc.

[0044]FIG. 19. Western blot using a monoclonal antibody tophosphorylated tyrosine.

DETAILED DESCRIPTION OF THE INVENTION

[0045] In some embodiments the invention provides methods and nucleicacids for expressing Clostridium botulinum genes in other prokaryotesand eukaryotes. More specifically, the invention provides methods andnucleic acids for expressing botulinum neurotoxin (BoNT) light chains(LC) in Escherichia coli or Pichia pastoris. In order to be expressed inEscherichia coli or Pichia pastoris, the sequence of DNA encodingwild-type BoNT LC is engineered to replace some Clostridium codons thatare rare or unrecognized in the host organism and to reduce the A+Tcontent. The recombinant or synthetic DNA molecules of the invention arepreferrably designed with codon usage normally found in genes that arehighly expressed in the host organism, e.g. Escherichia coli or Pichiapastoris. By selecting codons rich in G+C content, synthetic DNAmolecules may also be designed to lower the A+T-rich base compositionfound in the Clostridial genes. According to the invention, a host cellis a cell of any organism other than Clostridium. Nonlimiting examplesof host cells include gram negative bacteria, yeast, mammalian cells,and plant cells.

[0046] In some embodiments of the invention, upon expression of the DNA,a BoNT LC is produced in a heterologous host system by itself or as afusion with another protein or a carrier. Proteins with which BoNT LCsmay be fused include BoNT HCs, maltose-bonding proteins, otherneurotoxins, neuropeptides, and autofluorescent proteins. A syntheticlight chain gene may be genetically fused to a gene encoding a BoNT HC,producing recombinant botulinum toxin.

[0047] In some embodiments of the invention, BoNT LC is produced that is(i) substantially free of contaminating toxicity, (ii) moderately tohighly soluble in aqueous media, (iii) stable for at least about sixmonths at 4° C., (iv) catalytically active, (v) functionally active, orcombinations thereof. In some embodiments of the invention, gramquantities of BoNT LC may be obtained per liter of culture medium. Insome embodiments of the invention, a recombinant BoNT LC may reduce anyimmunological response that may result from the presence of bindingproteins associated with the recombinant BoNT LC.

[0048] In some embodiments, the invention provides BoNT LC thatsubstantially lacks catalytic activity as a zinc protease as measured bythe SNAP-25 assay described in Examples 8, 17, and, 25 below. In someembodiments, the invention provides nucleic acids that encoderecombinant BoNT LC substantially lacking catalytic activity as a zincprotease, wherein amino acids in or spatially near the active site aredeleted, replaced or modified relative to wild-type native BoNT.Catalytically inactive BoNT LC may be fused with BoNT HC to form amutant recombinant holotoxin. Such holotoxins may be used to carrymolecules, e.g., drugs, into cholinergic nerve cells.

[0049] In some embodiments, this invention provides a nucleic acidcomprising a nucleic acid sequence encoding the N-terminal portion of afull length botulinum neurotoxin (BoNT) selected from the groupconsisting of BoNT serotype A, BoNT serotype B, BoNT serotype C1, BoNTserotype D, BoNT serotype E, BoNT serotype F, and BoNT serotype G,wherein said nucleic acid is expressible in a recombinant organismselected from Escherichia coli and Pichia pastoris. In some preferredembodiments, the nucleic acid corresponds in length and encoded aminoacid sequence to the BoNT light chain (LC). In some particularlypreferred embodiments, the nucleic acid comprises a nucleic acidsequence selected from SEQ ID NO:4 (serotype A), SEQ ID NO:6 (serotypeB), SEQ Id NO:8 (serotype C1), SEQ ID NO:10 (serotype D), SEQ ID NO:12(serotype E), SEQ ID NO:14 (serotype F), SEQ ID NO:16 (serotype G), SEQID NO:22 (serotype B), SEQ Id NO:26 (serotype C1), SEQ ID NO:30(serotype D), SEQ ID NO:34 (serotype E), SEQ ID NO:38 (serotype F), andSEQ ID NO:42 (serotype G).

[0050] In preferred embodiments, nucleic acids of the invention aresynthetic nucleic acids. In some preferred embodiments, the sequence ofthe nucleic acid is designed by selecting at least a portion of thecodons encoding BoNT LC from codons preferred for expression in a hostorganism, which may be selected from gram negative bacteria, yeast, andmammalian cell lines; preferably, the host organism is Escherichia colior Pichia pastoris. The nucleic acid sequence encoding LC may bedesigned by replacing Clostridium codons with host organism codons thatencode the same amino acid, but have a higher G+C content. Conservativeamino acid substitutions are within the contemplation and scope of theinvention. In preferred embodiments of the invention, a nucleic acidencoding a recombinant BoNT or fragment thereof is capable of beingexpressed in a recombinant host organism with higher yield than a secondnucleic acid encoding substantially the same amino acid sequence, saidsecond nucleic acid fragment having the wild-type Clostridium botulinumnucleic acid sequence.

[0051] Codon usage tables for microorganisms have been published. Seee.g. Andersson S G E, Kurland C G, 1990, “Codon preferences infree-living microorganisms” Microbiol. Rev 54:198-210; Sreekrishna,1993, “Optimizing protein expression and secretion in Pichia pastoris”in Industrial Microorganisms: Basic and Applied Molecular Genetics,Baltz, Hegeman, Skatrud, eds, Washington D.C., p. 123; Makofl A J, OxerM D, Romanos M A, Fairweather N F, Ballantine S, 1989, “Expression oftetanus toxin fragment C in E. coli: high level expression by removingrare codons” Nuc. Acids Res. 17(24): 10191-10202. Table 3 ofSkreekrishna is a chart depicting codon usage in Pichia pastoris. Thistable was generated by listing the codons found in a number of highlyexpressed genes in P. pastoris. The codon data was obtained bysequencing the genes and then listing which codons were found in thegenes.

[0052] From such tables, it is clear that amino acid residues can beencoded by multiple codons. When constructing synthetic DNA moleculesusing P. pastoris codon usage, it is preferred to use only those codonsthat are found in naturally occurring genes of P. pastoris, and itshould be attempted to keep them in the same ratio found in the genes ofthe natural organism. When the clostridial gene has an overall A+Trichness of greater than 70% and A+T regions that have spikes of A+T of95% or higher, they have to be lowered for expression in expressionsystems like yeast. Preferably, the overall A+T richness is loweredbelow 60% and the A+T content in spikes is also lowered to 60% or below.In preferred embodiments of the invention, maintaining the same codonratio (e.g., for glycine GGG was not found, GGA was found 22% of thetime, GGT was found 74% of the time, GGC was found 3% of the time) isbalanced with reducing the high A+T content. In the construction of theDNA molecules of the invention, it is preferred to avoid spikes wherethe A+T content exceeds about 55%.

[0053] According to the invention, a spike may be a set of about 20 toabout 100 consecutive nucleotides. A spike having an high A+T contentgreater than 80% or 90% may function as transcription termination sitesin host systems, thereby interfering with expression. Preferredsynthetic DNA molecules of the invention are substantially free ofspikes of 50 consecutive nucleotides having an A+T content higher thanabout 75%. Alternatively, preferred synthetic DNA molecules of theinvention are substantially free of spikes of 75 consecutive nucleotideshaving an A+T content higher than about 70%. Alternatively, preferredsynthetic DNA molecules of the invention are substantially free ofspikes of 100 consecutive nucleotides having an A+T content higher thanabout 60%.

[0054] A synthetic DNA molecule of the invention designed by using E.coli codons is expressed fairly well in P. pastoris. Similarly, asynthetic gene using P. pastoris codons also appears to be expressedwell in E. coli.

[0055] In some embodiments, this invention provides an expression vectorcomprising a nucleic acid of this invention, whereby LC is produced upontransfection of a host organism with the expression vector. Anotherembodiment of this invention provides a method of preparing apolypeptide comprising the BoNT LC selected from the group consisting ofBoNT serotype A, BoNT serotype B, BoNT serotype C, BoNT serotype D, BoNTserotype E, BoNT serotype F, and BoNT serotype G, said method comprisingculturing a recombinant host organism transfected with an expressionvector of this invention under conditions wherein BoNT LC is expressed.Preferably, the recombinant host organism is a eukaryote. In anotherpreferred embodiment, the method of this invention further comprisesrecovering insoluble protein from the host organism, whereby a fractionenriched in BoNT LC is obtained. E. coli is a preferred host forexpressing catalytically-active (i.e., proteolytically-active) LC.Pichia pastoris is a preferred host organism for expressing inactive ormutated LC. Pichia pastoris has SNARE proteins which probably getinactivated by catalytically-active LC.

[0056] In some embodiments, the invention provides an immunogeniccomposition comprising a suitable carrier and a BoNT LC selected fromthe group consisting of BoNT serotype A, BoNT serotype B, BoNT serotypeC, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotypeG. Preferably, the immunogenic composition is prepared by culturing arecombinant organism transfected with an expression vector encoding BoNTLC. More preferably, the immunogenic composition is prepared by a methodwherein an insoluble protein fraction enriched in BoNT LC is recoveredfrom said recombinant organism. More preferably, the immunogeniccomposition is prepared by the method of Example 30.

[0057] According to some non-limiting embodiments, the inventionprovides reagents and compositions that are useful for developingtherapeutic interventions against BoNT. For example, the recombinantBoNT nucleic acids and polypeptides of the invention may be used toscreen for botulinum neurotoxin inhibitors.

[0058] In some embodiments, the invention provides therapeutic agentsfor clinical disorders such as dystonias, spasticity, and pain.According to these embodiments, the agents may be prepared by firstexpressing and purifying BoNT LC independently of any portion of theheavy chain. The BoNT LC so produced is then fused to the heavy chain orfragments thereof, e.g., HN and HC. Alternatively, BoNT LC may becoexpressed and/or copurified with BoNT HC or fragments thereof and thenfused to BoNT HC or fragments thereof. These agents may be used inclinical (human) or veterinary (non-human animal) applications.

[0059] In some embodiments, the invention provides agents that may beuseful for treating disorders associated with cholinergic nervefunction, SNAP-25, VAMP, syntaxin or combinations thereof. In someembodiments, the invention provides agents that may be useful forreducing any immunological response that may result from the presence ofbinding proteins associated with the agents. For example, the nativeBoNT holotoxin is highly immunogenic and some patients become refractoryto continued treatment with it over time as their protective antitoxintiter rises. The efficacy of holotoxin-based drugs (e.g., BOTOX,Myobloc/Neurobloc, Dysport) may be improved by pretreating patientshaving a high titer of anti-holotoxin antibodies with a holotoxinfragment such as Lc, Hn, or Hc. These fragments may bind theanti-holotoxin antibodies making them unavailable for binding thesubsequently administered holotoxin. This may work for a short time(months to a few years) realizing eventually that the antibody level maybe built up so much that the drug can no longer be effective even withthe addition of fragments. At this point in time, the patients will haveto use a different serotype toxin drug or a chimera of the toxin (i.e.,mixing toxin domains).

[0060] In further embodiments, the invention provides an immunogeniccomposition comprising a suitable carrier and a BoNT LC selected fromthe group consisting of BoNT serotype A, BoNT serotype B, BoNT serotypeC, BoNT serotype D, BoNT serotype E, BoNT serotype F, and BoNT serotypeG. Preferably, the immunogenic composition is prepared by culturing arecombinant organism transfected with an expression vector encoding BoNTLC. More preferably, the immunogenic composition is prepared by a methodwherein an insoluble protein fraction enriched in BoNT LC is recoveredfrom said recombinant organism.

[0061] The LC is present in immunogenic compositions of the invention inan amount sufficient to induce an immunogenic response thereto.

[0062] Two of the major advantages of the recombinant botulinumneurotoxins and fragements of the invention are the safety and highyields possible. First, the recombinantly-produced botulinum neurotoxin(rBoNT) protein fragments are completely nontoxic and are, thus, verysafe. The fermentation of the host cell harboring the rBoNT gene (e.g.,Escherichia coli or Pichia pastoris) does not require the highbiological containment facilities presently needed to ferment thespore-forming Clostridium botulinum required for the production of theneurotoxin light chains. Second, synthetic DNA molecules of theinvention can be placed in high expression systems and used to make muchlarger quantities of the BoNT fragments than toxin produced by theparent organism, Clostridium botulinum. Thus, there may be immense costsavings because it will be easier and safer to produce much largerquantities of the proteins for various uses including vaccination.

[0063] Synthetic DNA molecules as described herein may be transfectedinto suitable host organisms to create recombinant production organisms.Cultures of these recombinant organisms can then be used to producerecombinat BoNT fragments or holotoxins. Exemplary techniques fortransfection and production of BoNT fragments are shown in the Examples.Alternative techniques are well documented in the literature See, e.g.,Maniatis, Fritsch & Sambrook, “Molecular Cloning: A Laboratory Manual”(1982); Ausubel, “Current Protocols in Molecular Biology” (1991); “DNACloning: A Practical Approach,” Volumes I and II (D. N. Glover, ed.,1985); “Oligonucleotide Synthesis” (M. J. Gait, ed., 1984); “NucleicAcid Hybridization” (B. D. Hames & S. J. Higgins, eds., 1985);“Transcription and Translation” (B. D. Hames & S. J. Higgins, eds.,1984); “Animal Cell Culture” (R. I. Freshney, ed., 1986); “ImmobilizedCells and Enzymes” (IRL Press, 1986); B. Perbal, “A Practical Guide toMolecular Cloning” (1984), and Sambrook, et al., “Molecular Cloning: aLaboratory Manual” (1989). Such techniques are explained fully in theliterature. Modification of these techniques within the scope of thisinvention is within the skill in the art.

[0064] Recombinant forms of botulinum neutotoxin light chain may beuseful in one or more of the following applications: strabismus andother disorders of ocular motility, dystonia, blepharospasm, cervicaldystonia, oromandibular dystonia, laryngeal dystonia (spasmodicdysphonia), limb dystonia, hemifacial spasm and other facialdyskinesias, tremors of the head and hands, eyelid, cervical, and othertics, spasticity (e.g. anal), Stiff-Person syndrome, bladder dysfunction(e.g. in patients with spinal-cord injury), segmental myoclonus andother hyperkinetic disorders, cosmetic treatment of glabelar frown linesand other facial wrinkles, and all conditions characterized byhyperactivity of the lower motor neuron. See Cardoso and Jankovic, 1995and references cited therein. The light chain may further be used tocontrol autonomic nerve function (U.S. Pat. No. 5,766,605) ortiptoe-walking due to stiff muscles common in children with cerebralpalsy, according to findings published in the November 2001 issue ofPediatrics.

[0065] Absolute contraindications to the use of BoNT are allergy to thedrug and infection or inflammation at the proposed injection sitewhereas myasthenia gravis, Eaton-Lambert syndrome, motor neuron disease,and coagulopathy are relative contraindications (National Institutes OfHealth Consensus Development Conference Statement On Clinical Use OfBotulinum Toxin 1991; Report Of The Therapeutics And TechnologyAssessment Subcommittee Of The American Academy Of Neurology 1990).Safety for use during pregnancy and lactation has not been firmlyestablished (National Institutes Of Health Consensus DevelopmentConference Statement On Clinical Use Of Botulinum Toxin 1991).

[0066] The invention contemplates isoforms of the light chain as well aschimeras with other domains of the toxin or other proteins. In otherwords, gene fragments with DNA sequences and amino acid sequences notidentical to those disclosed herein may be discovered in nature orcreated in a laboratory. The invention contemplates the production ofany protein or polypeptide that has biological activity/functionalitysimilar to the wild-type botulinum neurotoxin light chain, e.g. cellbinding, translocation across membrane, catalytic activity sufficient toinactivate critical proteins in a cell involved with proteintrafficking, release of various chemical trasmitters (i.e.,acetylcholine, glutamate, etc.), hormones, etc.

[0067] For example, the light chain and translocation domain may becombined with a protein or peptide that targets a different receptorand/or cell-type. In addition, the invention contemplates therapeuticdelivery of synthetic DNA molecules of the invention to cells via viralvectors such as adenovirus or other gene therapy techniques.

EXAMPLES

[0068] In order to facilitate a more complete understanding of theinvention, a number of nonlimiting Examples are provided below forillustration purposes only. To advance these purposes, the Examples arearranged in four sets: Examples 1-13, Examples 14-20, Examples 21-29,and Example 30.

Example 1 Chemicals, Buffers, and Reagents

[0069] Buffer T (20 mM Tris-HCl, pH 9.2) and buffer G (50 mM sodiumglycine, pH 9.0) were used as indicated. SKL (sodium N-lauryl sarcosineor sarkosyl) was from Sigma. Highly purified (>95%) full-length BoNT/Awas purchased from List Biologicals (Campbell, Calif.). Rabbitpolyclonal antibodies against a 16-residue N-terminal sequence(PFVNKQFNYKDPVNGV; SEQ ID NO:1) of the BoNT/A LC were produced andaffinity purified by Research Genetics (Huntsville, Ala.).Peroxidase-coupled goat anti-rabbit and anti-mouse IgG (H+L) and ABTSsubstrate were from Kirkegaard Perry Laboratories (Gaithersburg, Md.).Oligonucleotides, designed for E. coli codon usage (Anderson andKurland, 1990) and ranging in size from 70 to 100 nucleotides, weresynthesized by Macromolecular Resources (Fort Collins, Colo.).

Example 2 Construction and Expression of a Synthetic DNA EncodingrBoNT/A LC

[0070] The DNA encoding the enzymatic LC domain of BoNT/A was assembledfrom three segments, a 335-base pair (bp) Sal I-Sph I fragment, a 600-bpSph I-Kpn I fragment, and a 460-bp Kpn I EcoR I fragment. To constructthe first segment, six oligonucleotide pairs were annealed, ligated,and, after PCR amplification, inserted into pGEM3Zf at Sal I-Sph Irestriction enzyme sites. The second segment was built by annealing andligating eight oligonucleotide pairs, followed by its amplification andinsertion into the Sph I and Kpn I sites of pGEM3Zf. The final segmentwas constructed by annealing and ligating six oligonucleotide pairs,followed by its amplification and insertion into the Kpn I-EcoR I sitesof pGEM3Zf. Nucleotide sequencing of gene fragments in pGEM3Zf wasperformed to identify clones in each group with minimalmisincorporations. In vitro mutagenesis was performed to correct themisincorporations in the BoNT/A LC minigene fragments. Directional geneassembly via 600-bp and 460-bp fragments in pGEM3Zf was followed by theinsertion of the 335-bp fragment.

[0071] In the design of the synthetic DNA, the 5′ oligonucleotide foramplifying the gene's 5′ terminus consisted of an anchored Sal I sitefollowed by an EcoR I site and an Nco I site to facilitate directionalsubcloning into the E. coli expression vector, pET24d. The 3′oligonucleotide contained a hexahistidine tag with a thrombin proteasecleavage site for creating a carboxyl-terminal removable histidine tag.The 3′ end also included the restriction enzyme sites for BamH I andEcoR I.

[0072] The full-length gene was excised from pGEM3Zf 5 with a Nco I-EcoRI and subcloned into a similarly digested pET24d vector. The resultingligated construct was used to transform E. coli BL21(DE3) cells. Twoclones were assayed for their ability to express rBoNTA LC. Singlecolonies were inoculated into 5 ml of Luria broth (LB) containing 50μg/ml of kanamycin and grown overnight at 37° C. The overnight cultures(500 μL) were used to inoculate 50 ml of LB containing 50 μg/ml ofkanamycin. When the cultures reached OD₆₀₀ of 0.8, induction wasinitiated by addition of isopropyl-β-D-thiogalactoside (IPTG) (finalconcentration, 1.0 mM). The cultures were induced for 2 hr at 37° C.,harvested, and analyzed for expressed products on SDS-PAGE.

Results

[0073] A synthetic DNA encoding rBoNTA LC was designed with E. colicodon usage, constructed, and expressed in E. coli. The native nucleicacid sequence from C. botulinum type A NTCC 2916 (Thompson et al., 1990)was used as the template for preparing synthetic LC sequences of theinvention.

[0074] At the 5′ end of the DNA, an Nco I restriction enzyme site wasemployed as a cloning site and palindrome to provide an initiationcodon. The use of this Nco I site necessitated the use of a filler codon(GTT) between the Met initiation codon (ATG) and the codon (CAG)specifying the first amino acid residue in the LC (i.e., Q). Thisresulted in the introduction of one extra amino acid, Val, as theN-terminal residue (after the initiating Met). This extra and new aminoacid, however, did not interfere with expression or activity (seelater). The length of the LC (448 residues) to be expressed was chosenfrom the sequence of amino acids around the nicking site (DasGupta andDekleva, 1990) (FIG. 1). At the C-terminal end (i.e., DKGYNK; residues444-449 of SEQ ID NO:5), a hexa-His tag was incorporated for affinitypurification and a thrombin cleavage site (LVPRGS; residues 450-455 ofSEQ ID NO:5) was incorporated for removing the hexa-His tag. Theexpressed protein therefore contained a total of 461 (1+448+6+6)residues (FIG. 1 and SEQ ID NO:5). The synthetic gene thus constructedin pET24d vector was highly and efficiently expressed in E. coli,accounting for about 25% of the total protein (FIG. 2).

Example 3 Fermentation

[0075] A frozen stock seed culture of recombinant E. coli harboring thesynthetic DNA encoding the LC of BoNT/A was grown at 37° C. to an OD₆₀₀of 2.682 in a shake flask containing 100 ml of the following definedmedium: casamino acids (1.4 g/L); yeast extract (2 g/L); (NH₄)₂SO₄ (1.85g/L); K₂HPO₄ (30 g/L); MgSO₄-7H₂O (2 g/L); thiamine.HCl (0.015 g/L);glucos (18.1 g/L); trace elements solution (3 ml/L) consisting ofFeCl₃.6H₂O, 27 g; ZnCl₂.4H₂O, 1.3 g, CoCl₂.H₂O, 2 g; Na₂Mo₄.2H₂O, 2 g;CaCl₂.2H₂O, 1 g; CuCl₂.2H₂O, 1 g; H₃BO₃, 0.5 g; distilled H₂O, 1000 ml;and HCl, 100 ml. In addition, 0.0156 g/L of ZnCl was added to traceminerals to make the concentration of Zn five times greater in the shakeflask and fermentor. Kanamycin (50 μg/L) was added as an antibiotic. Theshake flask culture was used to inoculate a 5-L BioFlo III fermentor(New Brunswick Scientific, Edison, N.J.) containing 4.3 L of the mediumdescribed above. Later in the growth (5.5 hr), 14.1 g/L of casaminoacids was added and a glucose feed was initiated to maintain a glucoseconcentration of 1 g/L. Growth continued for 8 hr until an OD₆₀₀ of 49.9was reached. Cell induction was then initiated at this time by addingIPTG (final concentration, 1.5 mM). Induction continued for 4 hr afteradding IPTG, and cells (OD₆₀₀ of 112.62) were harvested bycentrifugation (Beckman, Palo Alto, Calif.) at 7000 rpm for 15 min at 4°C. Cells were washed with cold 0.9% saline and centrifuged at 7000 rpmfor min and frozen at −70° C. Wet cell yield was 58 g/L.

Example 4 Extraction and Purification of Light Chain as Inclusion Bodies

[0076] In a typical preparation, 12 g of E. coli cells was suspended ina total volume of 30 ml of buffer T containing 5 mM MgCl₂, 1.5 mM PMSF,10 mM β-mercaptoethanol, and 2 mg of DNAse. The cell suspension wassubjected to 10 cycles of 2-min sonication (at 60% power in a FisherModel 300 Sonic Dismembrator) and 2-min cooling on ice. Aftercentrifugation for 15 min at 10,000 ×g, the supernatant was discarded.The pellet was suspended in 30 ml the above buffer. The cycle ofsonication and centrifugation was repeated five more times; MgCl₂ andDNAse were omitted from the buffer during the last two cycles. Theresulting pellet contained the rBoNT/A LC, that appeared ˜70% pure bySDS PAGE (FIG. 2). The pellet was stored at 4° C. as a white suspensionin 15 ml of buffer T containing 1.5 mM PMSF and 10 mM β-mercaptoethanol.

Results

[0077] The expressed LC appeared exclusively in the insoluble pelletfraction (FIG. 2). Including MgCl₂ and DNAse in the cell suspensionensured a clean separation of the pellet from the supernatant aftersonication and centrifugation. The white suspension of the purifiedBoNT/A LC migrated as a 52-kDa band and appeared to be ˜70% pure onSDS-PAGE (FIG. 2A), as determined by densitometric analysis. Minorcontaminant bands with ˜100-kDa, 37-40 kDa, and ˜25 kDa also reactedwith the antibody in the Western blot (FIG. 2B). While fragments smallerthan 50 kDa may have arisen from proteolysis of the LC (DasGupta andFoley, 1989), the origin of the 100-kDa species in the reducing SDS-PAGEgels is not clear since the species also reacts with theaffinity-purified antibodies against a small sequence of the LC.Molecular mass determination by MALDI-MS gave 52.774 (±50) kDa as thepredominant species along with minor species of 106.028 (±100) kDa and25.00 (±25) kDa. Amino acid sequence determination of the LC identifiedV-Q-F-V-N-K-Q as the amino-terminal sequence, as expected for theconstructed gene (FIG. 1) and identical (with the exception of thepenultimate valine) to that of the published sequence of BoNT/A(Thompson et al., 1990).

Example 5 Solubilization of the Inclusion Bodies to Obtain ActiverBoNT/A LC

[0078] In a typical experiment, 0.75 ml of the white rBoNT/A LCsuspension (from an equivalent of 600 mg of wet cells) was centrifugedin a 2-ml Eppendorf tube and the supernatant was discarded. The pelletwas suspended by mild sonication in 0.9 ml of 50 mM Tris-HCl, pH 9. A20% solution (0.9 ml) of SKL in water was added to the suspension atroom temperature and was mixed by inversion several times. Within 2 min,the pellet became completely soluble. Any remaining turbidity wascleared by further diluting with 50 mM Tris-HCl, pH 9.0, or was removedby centrifugation. The SKL-solubilized LC was dialyzed against 200volumes of buffer G containing 1 mM DTT with one to two daily changes at4° C. for 1 week. The yield of the soluble rBoNT/A LC was 12 mg (3.9mg/ml), which was stored in a glass tube at 4° C.

Results

[0079] The purified inclusion bodies were solubilized in 10% SKL and theSKL was removed by dialysis against buffer G containing 1 mM DTT (seeSection 2). The use of a 10% SKL solution ensured solubilization within2 min of incubation, and the LC solution was immediately subjected toextensive dialysis to remove the detergent. Starting with an equivalentof 600 mg of the wet E. coli cells, 12 mg of the soluble LC wasobtained, corresponding to 20 mg LC per gram of wet cells. Thiscorresponds to a yield of 1.16 g of the pure protein per liter of cellculture.

Example 6 Properties of the Purified BoNT/A LC

[0080] The UV-visible absorption spectrum (FIG. 3) shows the rBoNT/A LCwith a single maximum at 278 nm as a simple protein. Although a numberof minor band were observed in the SDS-PAGE gel (FIG. 2), absence of anyother absorbance bands in the UV-visible range suggests the absence ofany nonmetal cofactor in the preparation. The LC was expressed as aC-terminally His-tagged protein. In the presence of 6 M GuHCI, therBoNT/A LC was bound to Ni-resin and was eluted withimmiadzole-containing buffers as a more purified form. Without GuHCI,the rBoNT/A LC did not bind to Ni-resin. This result suggests that theLC retained the His-tag after expression and purification, but in theabsence of GuHCI, the His-tag was not exposed to solvent to chelate withthe Ni-resin. Because the rBoNT/A LC had catalytic properties comparableto those of the dicchain (see below), removal of the His-tag from thepurified protein was not attempted.

[0081] The purified LC was stable for at least 6 months when stored at4° C. in buffer G containing I mM DTT (FIG. 4A). During this period, theprotein remained fully soluble, did not show any degradation as analyzedSDS-PAGE, and retained its initial catalytic activity. An LC preparationobtained by prolonged solubilization in 0.5% SKL at room temperature,however, precipitated after 3 months of storage at 4° C. and lost mostof its initial catalytic activity. The LC (1 mg/ml of 50 mMNa-phosphate) precipitated from solution below pH 8 either at 4° C. orat 25° C. Thermal stability of the LC (3.74 mg/ml of buffer G containing1 mM DTT and 50 μM ZnCl₂) was investigated by incubating aliquots for 45min at various temperatures. After cooling on ice for 45 min, thecatalytic activities in the supernatants were measured. The midpoint ofthermal unfolding T_(m) as measured by activity was 43° C. (FIG. 4B). Atroom temperature, increasing concentration of MgCl₂ also precipitatedthe LC from solution: at 6 mM MgCl₂, >80% of the LC precipitated.

Example 7 Preparation of Apo-rBoNT/A LC

[0082] One milliliter of rBoNT/A LC (2.73 mg) was dialyzed overnightagainst 250 ml of buffer G containing 5 mM EDTA and 1 mM DTT. EDTA wasremoved by further dialysis for 60 hr against three changes of 250 ml ofbuffer G containing 1 mM DTT.

Example 8 Assay of Proteolytic Activity of BoNT/A LC

[0083] BoNT/A cleaves the glutamyl-arginine bond between residues 197and 198 of the 206-residue SNAP-25. Schmidt and Bostian (1995) showedthat a synthetic 17-residue peptide representing residues 187-203 ofSNAP-25 was sufficient for detecting endopeptidase activity of BONT/Aand allowing routine assay for the neuotoxin activity. The peptide thusprobably mimics the structure of SNAP-25 in vivo (Bi et al., 1995). Thesame peptide was used in an identical method to assay the proeolyticactivity of the BONT/A LC.

[0084] The assay is based on HPLC separation and measurement of thenicked products from a 17-residue C-terminal peptide of SNAP-25 (FIG.5), corresponding to residues 187-203, which is the minimum lengthrequired for BoNT/A proteolytic activity (Schmidt and Bostian, 1995,1997). Unless otherwise noted, a 0.03-ml assay mixture containing0.8-1.0 mM substrate, 0.25 mM ZnCl₂, 5.0 mM DTT, 50 mM Na-HEPES buffer(pH 7.4), and BONT/A LC was incubated at 37° C. for 15-80 min. Theamounts of uncleaved substrate and the products were measured afterseparation by reverse-phase HPLC (Waters) on a Hi-Pore C 18 column,0.45×25 cm (Bio-Rad Laboratories, Hercules, Calif.) with the Millenniumsoftware (Waters) package. Solvent A was 0.1% TFA and solvent B was 70%acetonitrile/0. 1% TFA. The flow rate was 1.0 ml/min at 25° C. After thecolumn was equilibrated with 10% B, the sample was injected, and thecolumn was held at 10% B for 2.5 min. A linear gradient to 36% B over 21min was followed by 100% B for 6 min. Kinetic parameters for thesynthetic substrate were calculated from Lineweaver-Burk plots ofactivity with peptide concentrations from 0.26 to 1.7 mM.

Catalytic Activity of the LC

[0085] The BoNT/A LC is zinc-endopeptidase specific for the cleaving thepeptide bond between residues 197 (Glu) to and 198 (Arg) of SNAP-25.Incubating the 17-mer synthetic peptide representing residues 187-203 ofSNAP-25 with the LC at 37° C. for 5-200 min generated only two peptides(FIG. 5). That no other peptide fragments were generated by thisprolonged incubation proves that the contaminants present in the LCpreparation were devoid of any proteolytic activity. Incubating the LCwith BSA also failed to produce any proteolytic fragment. In contrast tothe BoNT/A dichain, whose activity ruin is greatly enhanced by BSA(Schmidt and Bostian, 1997), the rate of cleavage of the syntheticpeptide substrate was unaffected by the presence of BSA.

[0086] Proteolytic activity of the purified rBoNT/A LC linearlyincreased with the increasing amount of the LC in the reaction mixture.The time course of activity (at 0.8-1.0 mM substrate concentration),however, was not linear, but progressively declined, possibly due to ahigh K_(m) for the substrate peptide (see below). Therefore, routineassays depended on initial activities representing <30% substrateconversion.

[0087] Substrate K_(m) for the LC was fourfold lower than that reportedfor the dichain (Schmidt and Bostian, 1995). This may be due toshielding of the active site by a ‘belt’ from the translocation domain(H_(n)) in the dichain neurotoxin (Lacy et al., 1998; Lacy and Stevens,1999). Thus, the ‘belt’ may pose a steric hindrance for substratebinding by the dichain (high K_(m)).

[0088] Nonetheless, the catalytic efficiency k_(cat)/K_(m) of the freerBoNT/A LC was somewhat higher than that of he dichain.

Optimum pH, Salts, and Buffers

[0089] An optimum pH of 7.2 for the proteolysis of the syntheticsubstrate by the rBoNT/A LC was determined by assaying in threedifferent buffer systems (0.1 M) ranging in pH from 5.0 to 9.0 (FIG. 6).For comparison, the optimum pH values of BoNT/B and tetanus neurotoxin,two members of the clostridial neurotoxin family, are 6.5-7.0, and6.5-7.5, respectively (Foran et al., 1994). Tris-HCI appeared to have aninhibitory effect on proteolysis, presumably due to chelation with thezinc at the active site. The activity at pH 7.4 was 25% higher in a 50mM HEPES buffer than in 100 mM HEPES. Adding 50 mM NaCl, KC1, or NaPO₄(pH 7.4) to the standard reaction mixture reduced activity 40-50%. Thus,high salt concentrations inhibited the proteolytic reaction.

Effect of Metals and ThiolReagents on Activity

[0090] BONT/A LC is a zone-endopeptidase. Activity of the rBoNT/A LC wascompletely inhibited by including the metal chelator EDTA (1 mM) in thereaction mixture (Table 1). Adding low concentrations of ZnCl₂ (1-50 μM)in the assay mixture slightly stimulated the activity (5%-10%) andhigher concentrations of ZnCl₂ inhibited the activity (FIG. 7). Theresults suggest that the active site should be almost saturated withZn²⁺ for optimum activity. The metal was tightly bound to the activesite of the LC, as the extraction, purification, or dialysis bufferswere devoid of Zn²⁺. Like Zn²⁺, other divalent metal ions, notably,MnCl₂ and NiSO₄, also inhibited the LC reaction to various extents inthe absence of added thiol (Table 1). Addin 5 mM DTT to the reactionmixture neutralized the inhibitory effect of Zn²⁺ (FIG. 7).

[0091] Neurotoxic or proteolytic activity of the dichain BONT/A probablyrequires an initial reduction of the disulfide bond between the LC andthe HC (de Paiva et al., 1993). Therefore, the proteolytic assay mixtureof BONT/A with the synthetic or natural substrates were supplementedwith 5-10 mM DTT (Washbourne et al., 1997; Schmidt and Bostian, 1995,1997). In the absence of Zn²⁺, 5 mM DTT in the reaction mixturesignificantly inhibited the activity of the LC (Table 1 and FIG. 7).Similarly, L-cys, dithioerythreitol, and glutathione inhibited theactivity to various extents, while β-mercaptoethanol stimulated theactivity in the absence of added Zn²⁺. These results were unexpected asthe LC does not possess any disulfide bonds and the invariant Cysresponsible for the interchain disulfide is far from the active site.One explanation for these effects is the formation of a mixed disulfidebetween a protein thiol and the exogenous thiol. To investigate theimportance of a protein Cys residue on activity, several sulfhydrylreagents were incubated in the proteolytic assay mixture (Table 1). BothHgCl₂ and p-Cl-mercuric benzoate completely abolished the activity ofLC. Preincubating the LC with these two reagents, then diluting with theproteolytic reaction mixture, also gave the same results. These resultssuggest the presence of a protein thiol in the vicinity of the activesite of the LC. TABLE 1 Effect of Metal Ions and Thiols and ThiolReagents on the Activity of the rBoNT/A LC Concentration MetalConcentration Thiol reagent (mM) % Activity reagent (mM) % ActivityNone^(a) 100 EDTA 1 00 Dithiothreitol 5 45 ZnCl₂ 0.25 60Dithioerythreitol 5 60 — 1 10 β-Mercaptoethanol 5 120 — 0.25Glutathione, reduced 5 75 +Dithiothreitol 5 125 Glutathione, oxidixed 575 MnCl₂ 1 40 S-Nitrosoglutathione 5 55 MgCl₂ 1 90 L-Cysteine 5 20 CaCl₂1 75 p-C1-Mercuribenzoate 0.050 00 FeCl₃ 1 35 Mercuric chloride 0.013 00CoCl₂ 1 90 Iodoacetamide 10 80 CuSO₄ 1 95 NiSO₄ 1 55

Steady-State Kinetic Parameters

[0092] The dependence of reaction rates on the substrate concentrationwas determined at 0.26-1.7 mM substrate at pH 7.4. A double reciprocalplot of the reaction rates versus substrate concentrations (FIG. 8)yielded a K_(m) of 1.18 mM and a V_(max) of 1670 (equivalent to 2390considering a 70% pure LC) nmol/min/mg LC (k_(cat)=1.39/sec or 1.99 if70% pure). For comparison, the maximum rate of cleavage of the peptidesubstrate by the native, dichain toxin is reported to be 1900nmol/min/mg (k_(cat)=4.7/sec), while the K_(m) is 5 mM (Schmidt andBostian, 1997). The lower K_(m) for the LC may be due to a more exposedactive site in the free LC than in the LC of the dichain, where theactive site is shielded from the solvent by elements of themembrane-spanning domain H_(N) (28-29). The catalytic efficiencyk_(cat)/K_(m) of the rBoNT/A LC, 1.18(1.69 if 70% pure), is thus higherthan that of the dichain, 0.94 (Schmidt and Bostian, 1995, 1997).

Apo-BoNT/A LC

[0093] The rBoNT/A LC was incubated with the metal chelator EDTA andafter extensive dialysis, the activity of the apo-BONT/A LC was measuredin the standard reaction mixture. In the absence of any exogenous Zn²⁺or thiol, the preparation had 17% activity of the holo-BONT/A LC fromwhich the apoprotein was made (Table 2). This result suggests that thebound Zn²⁺ was not completely removed by the EDTA treatment anddialysis. Nonetheless, adding 5 mM DTT and 250 μM ZnCl₂ to the assaymixture restored 70% of the activity of the holo-LC. Moreover, in thepresence of 5 mM DTT and 250 μM MnCl₂, MgCl₂, or CaCl₂, 20-30% of theoriginal activity was restored. TABLE 2 Activities of the Apo-BoNT/A LCWith and Without Addition of Divalent Metal Ions to the ReactionMixtures LC form Divalent metal % Activity % Activity recovered^(a)Holo-LC +Zn²⁺ 100  — Apo-LC +None 15 — +Zn²⁺ 70 65 +Mn²⁺ 20 10 +Mg²⁺ 2010 +Ca²⁺ 30 20 +Fe²⁺  0 —

Example 9 Vaccination of Animals

[0094] Purified rBoNTA LC was tested for its ability to elicitprotective immunity in Cr1:CD-1 (ICR) male mice (Charles River) weighing16-22 g. Two concentrations of recombinant LC (5 and 15 □g) with andwith-out adsorption to a 0.2% Alhydrogel (Superfos Biosector, Kvisgaard,Denmark) were administered in 0.9% saline in a total volume of 100 μl.Groups of 10 mice including a naive control (saline alone) receivedthree doses of LC at 0, 2, and 4 weeks. Mice were bled from theretroorbital sinus 12 days postvaccination and their antibodies assayedfor titers to toxin. Animals were challenged with native BoNT/A dichaintoxin 15 days postvaccination.

[0095] The animal room was maintained at 21±2° C. with a relativehumidity 30-70%, a 12/12-hr light/dark cycle with no twilight, and 10-15air changes/hour. Mice were housed in solid-bottom, polycarbonateMicro-Isolator™ cages (Lab Products, Inc., Seaford, Del.) with paperchip bedding (Alpha-Dri™, Shepherd Specialty Papers, Inc., Kalamazoo,Mich.) and provided food (Harlan Teklad diet No. 7022, NIH-07) and waterad libitum. All procedures were reviewed and approved by theInstitutional Animal Care and Use Committee and performed in an AAALACInternational-accredited facility in accordance with recommendations inthe Guide for the Care and Use of Laboratory Animals, 1996 (NationalAcademy Press, National Academy of Sciences, Washington, D.C.).

Example 10 ELISA

[0096] Highly purified (>95%) BoNT/A toxin was diluted to 2 μg/ml inphosphate-buffered saline (PBS), pH 7.4 (Sigma Chemical Co., St Louis,Mo.) and was dispensed (100 μl/well) into microtiter plates (Immulon 2,Dynatech Laboratories, Chantilly, Va.). The plates were incubatedovernight in a humidity box at 40° C. Five percent skim milk (Difco,Detroit, Mich.) in PBS with 0.01% Thimerosal® was used to blocknonspecific binding and as an antibody diluent. The plates were washedwith PBS plus 0.1% Tween 20 between each step. Mouse sera were initiallydiluted 1:100 and then diluted fourfold for a total of eight dilutions(1:100 to 1:1,600,000). Diluted sera were added in duplicate totoxin-coated wells (100 μl/well). The secondary antibody was horseradishperoxidase-conjugated, goat anti-mouse IgG diluted 1:1000. The primaryand secondary antibodies were incubated 90 and 60 min, respectively at37° C. ABTS substrate (100 μl/well) was added as the color developer.The plates were incubated at room temperature for 30 min. The absorbancewas measured with a microplate reader at 405 nm. A mouse monoclonalantibody, 5BA2.3, was used as the positive control in each assay; naivemouse serum was added as a negative control in each assay. The titer wasdefined as the geometric mean of the ELISA titer to BoNT/A toxin.

Example 11 Biological Effects of the rBoNT/A LC

[0097] LC prepared from dichain BoNTs always had residual toxicity dueto some contaminating dichain forms (Maisey et al., 1988). Todemonstrate and confirm that the rBoNT/A LC was nontoxic, 5-15 μg of theLC was injected per mouse, a dose that was 15,000-45,000 times higherthan an equivalent lethal dose of the BoNT/A dichain. Table 3 shows thatall the mice survived three successive injections. All of their antiserahad high titers against BoNT/A, but these antibodies failed to protectthe animals upon subsequent challenge with relatively low doses (10²LD₅₀) of the toxic BoNT/A dichain. Even when the ELISA titers wereboosted 20-fold by using the aluminum hydroxide adjuvant, the animalswere not immune to modest levels of BoNT/A challenge (Table 3).Comparable vaccination with BoNT/A Hc protected animals from challengewith as high as 1⁶ LD₅₀ (Smith, 1998). These results clearly demonstratethat the rBoNT/A LC was nontoxic to the animals and confirms earlierobservations that LC does not possess any neutralizing epitope(s) (Chenet al., 1997; Dertzbaugh and West, 1996). TABLE 3 Survival of Mice AfterVaccination with the rBoNT/A LC and Subsequent Challenge by BoNT/ADichain Survival at given Dose^(a) BoNT/A dichain challenge^(c)(μg/mouse) ELISA Titer^(b) 10²LD₅₀ 10³LD₅₀  0^(d)   <100 0/5  0/5  5^(d) 18,000 0/10 0/10 15^(d) 63,100 0/10 0/10  0^(e)   <100 0/5  0/5  5^(e)   985 0/10 0/10 15^(e)   2800 0/10 0/10

[0098] Although the LC by itself is nontoxic, in digitonin-permeabilizedchromaffin cells (Bittner et al, 1989) and direct microinjection intothe cytosol of sea urchin eggs (Bi et al., 1995; Steinhardt et al.,1994), it blocks membrane exocytosis. To demonstrate that the rBoNT/A LCpreparation retained this property of inhibiting membrane exocytosis,sea urchin eggs were microinjected with the LC. Eggs of the sea urchin,Lytechinus pictus, are an excellent model system for the study ofexocytosis. Unfertilized eggs have a layer of vesicles, the corticalgranules, docked at the plasma membrane. The SNARE complexes of dockedvesicles are inaccessible to the BoNTs. Thus, plasma membrane resealingof the unfertilized sea urchin egg is unaffected by microinjection withbotulinum toxins A, B, and C1 (Bi et al., 1995; Steinhardt et al.,1994). Fertilization triggers exocytosis of the cortical granuoles.After fertilization, the vesicles available for exocytosis are largelyundocked and the docking proteins of undocked vesicles are susceptibleto proteolysis by injected clostridial neurotoxins.

[0099] For fertilized eggs injected with rBoNT/A LC, about 100 min at20° C. was required to inhibit plasma membrane resealing aftermechanical wounding with a glass micropipet. Eggs that successfullyresealed showed a transient dye loss for about 1-2 min aftermicropuncture. Eggs that failed to reseal continuously lost dye and lostcontrol of intracellular free calcium, leading to cell death. Five offive fertilized eggs wounded between 36 and 70 min after injection withthe rBoNT/A LC resealed successfully, as did five of five unfertilizedinjected eggs. Six of six fertilized eggs wounded between 106 and 145min after injection failed to reseal, indicating that the recombinantlight chain actively inhibited exocytosis. Thus, the rBoNT/A LC had asimilar effect as BoNT/B in inhibiting membrane exocytosis and resealingof plasma membrane of sea urchin eggs (Steinhardt et al., 1994).

Example 12 Exocytosis Experiments

[0100] Plasma membrane resealing after micropuncture with a glasspipette requires calcium-regulated exocytosis (Bi et al., 1995). Thisexocytosis is dependent on docking proteins (the SNARE complex) that aresensitive to proteolysis by the clostridial neurotoxins (Steinhardt etal., 1994). Sea urchin (Lytechinus pictus) eggs were used to test thebiological activity of the rBoNT/A LC. The microinjection mediumcontained 19 volumes of the rBoNT/A LC (3.7 mg/ml) in 45 mM potassiumaspartate, 5 mM HEPES, pH 8.1, and one volume of 55 mM fura-2 in 100 mMKCl and 10 mM HEPES, pH 7.1. Injection levels were 5-10% of egg volume.The plasma membrane resealing after micropuncture with a glass pipettewas monitored by recording the emission from fura-2 upon excitation at358 nm (the calcium-insensitive wave-length).

Example 13 Other Analytical Methods

[0101] Protein concentration was determined by BCA assay (Pierce) withbovine serum albumin (BSA) as a standard. Reducing SDS-PAGE with 10%tricine-gels (Novex) was according to Laemli (1970). The gels werestained with Coomassie brilliant blue. Western blots were prepared byusing a primary polyclonal antibody against a 16-residue N-terminalsequence of BONT/A LC and a peroxidase-coupled goat anti-rabbit IgG(H+L) as the secondary antibody. Absorption spectrum at 25° C. wasrecorded in a Hewlett-Packard 8452 diode array spectrophotometer. TheN-terminal amino acid sequence of the BONT/A LC was determined by Edmandegradation in an Applied Biosystems Procise Sequencer in the 0- to20-pmol detection range. Molecular mass was determined by MALDI-MS in aPE Biosystems Voyager DE instrument. Sinapinic acid was used as thematrix and the sample was spotted on a stainless steel plate that wasnot washed with water or TFA. Other conditions in the experiment wereaccelerating voltage 25,000 V, guide wire voltage 0.3%, and laser 2500.

Example 14 Chemicals, Buffers and Reagents

[0102] Buffer P (50 mM Na-phosphate, ph 6.5) was used for Examples14-20. TPEN and ZnCl₂ were from Sigma. Affinity-purified,peroxidase-coupled goat anti-rabbit and anti-mouse IgG (H+L) and ABTSsubstrate were from Kirkegaard Perry Laboratories (Gaithersburg, Md.).The inhibitor peptide (Ac-CRATKML-NH₂) (Schmitd et al, 1998) wassynthesized and purified by Cell Essentials (Boston, Mass.).

Example 15 BoNT/A LC Purification

[0103] The rBONT/A LC was expressed by low-temperature IPTG induction inE. coli BL21 (DE3) cells as a soluble protein from a synthetic gene in apET24a-derived multicopy plasmid (Clontech, Inc.). Construction of thegene and expression of the protein as described (Ahmed and Smith, 2000)was modified as follows: a stop condon replaced the histidine tag at thecarboxy terminus of the gene, and induction and expression was at 18° C.for 22-24 hr. The LC was purified to near homogeneity by NaCl gradientelution from each of two successive cation exchange columns (MonoS) inbuffer P. Details of the expression and purification will be publishedelsewhere (manuscript in preparation). A typical preparation had aspecific activity of 2-3 μmol/min/mg in cleaving the 17-residuesubstrate peptide (see later) when assayed in the presence of 0.25 mMZnCl₂; in the absence of added zinc, activity was 50%. The purified LCwas thus partially resolved of the bound zinc. The purified protein (1-4ml) in buffer P was stored at −20° C. Under this condition, the proteinremains stable and retains its catalytic activity for at least 1 year.

Example 16 SDS-PAGE, Transfer on PVDF Membrane, and Western Blot

[0104] SDS-PAGE under reducing conditions (Laemmli, 1970) was carriedout on a 1-mm-thick 10% tricine gels (Novex) as described (Schagger andvon Jagow, 1987). Samples were prepared in 0.4% SDS, 5%β-mercaptoethanol, 12% glycerol, and 450 mM Tris-HCl, ph 8.45, byboiling for 5 min. The running buffer contained 0.1% SDS in 0.1 MTris-0.1 M Tricine, ph 8.3. The gels were stained with CoomassieBrilliant Blue. Electrophoretic transfer of peptides from SDS-PAGE gelsonto PVDF membrane used 10 mM CAPS-NaOH buffer, Ph 11.0, containing 10%methanol as the transfer buffer. Protein bands on the PVDF membraneswere visualized by 1 min of staining with Coomassie Brilliant Bluefollowed by destaining in 10% acetic acid-5% methanol. The stained bandswere cut out from the dried membranes for amino-terminal sequencedetermination. Western blots on nitro-cellulose membranes were preparedusing a primary polyclonal antibody against a 16-residue N-terminalsequence of BoNT/A LC and a peroxidase-coupled goat anti-rabbit IgG(H+L) as the secondary antibody (Ahmed and Smith, 2000).

Example 17 Proteolysis Experiments

[0105] Before each experiment, aliquots of the protein were thawed toroom temperature and were immediately passed through a PD-10 column toremove the EDTA. The protein was collected in buffer P and stored onice. The EDTA-free BoNT/A LC was mixed with predetermined concentrationsof ZnCl₂, EDTA, TPEN, or the inhibitor peptide (see later) and 20-50 μLwas distributed in screw-capped Eppendorf tubes. The tubes wereincubated at 4° C. or at 22° C. The final concentration of the proteinwas 0.18-0.20 mg/ml in these incubation mixtures. At various timeintervals an equal volume (20-50 μl) of SDS-load buffer was added to atube for SDS-PAGE analysis.

[0106] A 100 mM stock solution of TPEN was prepared in ethanol (95%).Stock solutions of the competitive inhibitor peptide Ac-CRATKML-NH₂(Schmidt et al., 1998) (5 mM), ZnCl₂ (1-4 mM), and EDTA (20 mM) wereprepared in buffer P. Unless otherwise mentioned, final concentrationsof these reagents in the incubation mixtures with the LC were TPEN 5 mM,EDTA 5 mM, peptide 1 mM, and ZnCl₂ 0.25 mM.

Results: Cleavage and Fragmentation of BoNT/A LC

[0107]FIG. 10 shows that the BoNT/A LC undergoes cleavage andfragmentation that increases with time. The intensity of the bandrepresenting the full-length LC with a polypeptide mass of ˜52 kDa (IA)gradually diminished with time and a new protein band of ˜50 kDa (IB)appeared in its place. The results suggest truncation of about 2 kDamass from the full-length LC. In Western blots (FIG. 10B), both IA andIB also reacted with a rabbit polyclonal antibody raised against a16-residue amino-terminal sequence of LC. This result suggests that thetruncation from the full-length LC must occur at the C-terminus. Indeed,amino-terminal sequencing of the isolated, truncated protein (see later)showed the amino terminus was intact. Interestingly, preservation of theN-terminus of full-length BoNT/A neurotoxin was also observed after itsposttranslation modification in bacterial culture (DasGupta and Dekleva,1990). As the truncated protein IB accumulated, a protein band of ˜100kDa (II) appeared that was detected easily in the Western blot (FIG.10B). FIG. 10 also shows that at 2 weeks of incubation, the LCfragmented into IIIA+IIIB and IVC. The larger fragment (IIIA) above the34-kDa marker was followed by a fainter fragment (IIIB) just below the34-kDa marker. The results of this time course experiment also suggestedthat IIIB was formed from IIIA. Both of these fragments must representthe N-terminus of the LC, as they reacted with the antibody (FIG. 10B).On the other hand, a much smaller fragment (IVC) moving faster than the23-kDa marker was probably the C-terminal fragment, as it failed toreact with the antibody (specific for the N-terminus of the LC) in theWestern blot. The truncation and fragmentation shown in FIG. 10 wereindependent of the batch of E. coli cell culture or the batch ofpurification of the LC.

Results: Zinc Accelerates the Truncation and Fragmentation

[0108] The BoNT/A LC is known to be highly substrate specific.Therefore, the truncation of about 2 kDa from the C-terminus orfragmentation into larger fragments upon storage of the LC at 4° C.described in FIG. 10 might appear to be due to the presence of somecontaminating protease in the LC preparation. However, no additionalCoomassie-stained protein bands were detected when 0.4-4.0 μg of the LCwas electrophoresed in the presence of SDS. BoNT/A LC is azinc-endopeptidase. FIG. 11 shows that when LC was incubated with 0.25mM ZnCl₂, the rate of fragmentation was greatly increased so that theantibody-reacting fragment IIIB and an antibody-nonreacting fragment IVAappeared within 2 days of incubation (FIG. 11A, B). Fragment IVBappeared later in the time course. Qualitatively, the results aresimilar to those in FIG. 10 except that in the presence of ZnCl₂, therate of fragmentation was higher, fragment IIIB was formed withoutshowing the initial formation of IIIA, and initial formation of IVA gaverise to IVB. The rate enhancement by zinc could be partly due toformation of holo-LC from the partially Zn-resolved LC (see Section 2).Because there was no fragment IVC (FIG. 10) detected in this experiment(FIG. 11), zinc must also have a structural role in the LC. From theresults shown in FIG. 11A it is not possible to judge if the C-terminaltruncation of IA in forming IB and dimerization in forming II precedethe fragmentation into III and IV. However, in some other experiments,using a lower concentration of ZnCl₂, it was possible to show thatformation of IIIB occurred before formation of IB and that fragmentationwas the last event.

[0109] The rates of C-terminal truncation and fragmentation of LC eitherin the absence or in the presence of ZnCl₂ were much higher whenincubated at 22° C. than at 4° C. In fact, amino-terminal sequence wasdetermined on the fragments generated by incubation at 22° C. for 2 daysonly (see later).

Results: Metal Chelator TPEN Inhibits Truncation and Fragmentation

[0110] As shown in FIG. 11, if the C-terminal truncation andfragmentation of the LC was indeed dependent on the presence of zinc,removing zinc from the incubation mixture and from the active site ofthe LC would be expected to abolish the truncation and fragmentationevents. However, zinc is very tightly bound to the active site of LC.Extensive treatment with 10 mM EDTA in the cold (Ahmed and Smith, 2000)or with 10 mM EDTA at room temperature (Li and Singh, 2000) failed tocompletely remove zinc from the active site of the LC. In agreement withthese observations, including 10 mM EDTA failed to protect the LC fromC-terminal truncation and processing (FIG. 12A). In contrast, the metalchelator TPEN largely protected the LC from truncation and fragmentation(FIG. 12A). It was also found that, at 1 mM TPEN, the LC showed noactivity when assayed for 5 min. Because the incubation mixture withTPEN did not contain any exogenous metal or zinc, any chelation by TPENmust have involved the active-site zinc of the LC. These results alsosuggest that truncation and fragmentation of the LC upon storage 4° C.or at room temperature were autocatalytic.

Example 18 Separation of Peptides with HPLC and Their Characterizationby ESIMS-MS

[0111] For mass and sequence determination, peptides were separated onan Agilent Technologies Series 1100 liquid chromatograph with a 0.8×100mm Poros-2 R/H column (PerSeptive Biosystems, Inc.). The mobile phasewas 0.1% formic acid (solvent A) and 80% acetonitrile in 0.1% formicacid (solvent B). The peptides were eluted with a linear gradient of0-100% B over 15 min at a flow rate of 0.2 ml/min. The injection volumewas 10 μl. The peptides were detected and structurally characterized ona Finnigan LCQ Deca mass spectrometer employing data-dependent MS/MS.Molecular mass was also determined by MALDI-MS with a PE BiosystemsVoyager DE instrument. Sinapinic acid was used as the matrix, and thesample was spotted on a stainless steel plate that was not washed withwater or TFA. Other conditions in the experiment were acceleratingvoltage 25,000 V, guide wire voltage 0.3%, and laser 2500.

Results: Amino Acid Sequence of the Small Peptides Generated byC-Terminal Processing

[0112] To map the sites of proteolysis, the small peptides were isolatedby ultrafiltration of a C-terminally truncated LC mixture. Amino acidsequences of these peptides were determined by ESIMS-MS (Table 4). Thepeptides with G433 at the amino terminus (peptide 4) and K438 at thecarboxy terminus (peptide 5) indicated cleavage by a trypsin-likeprotease on the R432-G433 and K438-T439 bonds, respectively. Of these,only the lysyl bond at K438 was reported to be cleaved by a clostridialendogenous protease or by trypsin (DasGupta and Dekleva, 1990). However,a cleavage at the K444-G445 bond as reported before by an endogenousclostridial protease (DasGupta and Dekleva, 1990) was not detected.Neither was cleavage detected at K440-S441 or at K427-L428 bonds, theother potential sites of tryptic cleavage. Although these resultsindicated that the LC preparations did not contain a protease activitythat could cleave at K427-L428, K440-S441, and K444-G445, it is equallypossible that some of the small peptides generated by cleavage at thesesites were lost during sample preparation. Interesting findings of thisexperiment (Table 4) are the peptides with N-terminus of T420(peptide 1) and V431 (peptide 3), as the preceding residues at F419-T420and C430-V431 bonds, respectively, are certainly not the sites of“tryptic” cleavage. TABLE 4 C-Terminal Peptides Generated after InitialCleavage of the BoNT/A LC^(a)    420  425  430  435  440  445   |    |    |    |    |    | Peptide Mass^(b)KNFTGLFEFYKLLCVRGIITSKTKSLDKGYNK^(c) 1 2188 (2188)   TGLFEFYKLLCVRGIITSK 2 2124 (2112)^(d)             CVRGIITSKTKSLDKGYNK^(d) 3 2008 (2008)              VRGIITSKTKSLDKGYNK 4 1753 (1753)                GIITSKTKSLDKGYNK 5  989 (977)^(d)             CVRGIITSK^(d) # the LC (Ahmed and Smith, 2000).

[0113] The sequence data from the ESIMS-MS results for the peptides 2and 5 agree very well with the residue stretches V432-K449 and with theresidue stretches V432 -K449 and with the residue stretches V431-K438,respectively. However the experimentally determined mass for “C430”, theresidue at the amino side of V431 in both peptides, was greater by 12.1Dalton than the theoretical mass for cysteine. At this stage, there issome uncertainty regarding the discrepancy in the mass of this“cysteine.” Chemical modification experiments using iodoacetamide oracidified methanol failed to shift the masses of these peptides,indicating that the suspected “cysteine” did not have a free sulfhydrylgroup nor was a contaminating aspartic acid. Cysteine in proteins areknown to occur as derivatives such as cysteine sulfenic acids (Ahmad andClaiborne, 1992; Claiborne et al., 1999). Attempts are being made todecipher the chemical nature of this “cysteine.” If indeed it was amodified C430, cleavages at the carboxy ends of F419, C430, and V431 inaddition to R432, K438, and K438 indicate that the proteolytic activityin this preparation was not “tryptic” in nature, but had a broadspecificity.

Results: Identity of the Large Peptides Generated by Fragmentation

[0114] The large peptides generated by fragmentation in the middle ofthe LC were identified by comparing the mass determined by MS with acalculated mass for a stretch of sequence based on the amino-terminalsequence determination (Table 5). Agreements between the experimentaland calculated values were within 0.07%. Identity of IIIA as having asequence range of V1-F266 was based on the kinetics of its (and ofIVC's) appearance on SDS-PAGE (FIGS. 10 and 11) and N-terminal sequenceof IVC. The sequence data along with Western blot results clearlydemonstrated that the amino terminus of the LC (IA and IB) remainedunchanged during the prolonged incubation period. Although theC-terminal sequence of the peptides IIIA and IIIB was not determined,N-terminal sequences of the peptides IVA, IVB, and IVC (Table 5)indicate that fragmentation of IA and IB (FIGS. 10 and 11) occurred bycleavage at the Y250-Y251 and F266-G267 bonds. Again, if the cleavagesof these tyrosyl and phenylalanyl bonds were catalyzed by a protease, itmust have been “nontryptic” in nature. Identity of the peptides IVB andIVC as having F423 at the C-terminal indicated that a C-terminalprocessing of the LC at F423-E424 remained undetected in the smallpeptide isolation experiment (see previous section). This resultnonetheless supports that C-terminal processing occurred at phenylalanylbonds in addition to lysyl, arginyl, valyl, and (most likely) cysteinylbonds. TABLE 5 Identity of the Polypeptides Generated by Proteolysis ofthe BoNT/A LC Mass Mass Sequence N-terminal Peptide^(a) (Exp) (Calc)range sequence IA 51,315 51,318  V1-K448 2-VQFVNKQ IB 48,866 48,870 V1-Y426 2-VQFVNKQ II 97,727^(b) 97,870^(b) IIIA n.d.^(c) 32,270 V1-F266 2-VQFVNKQ IIIB 28,111 28,130  V1-Y251 2-VQFVNKQ IVA 23,20723,207 Y252-K448 252-YEMSGLE IVB 20,319 20,319 Y252-F423 252-YEMSGLE IVC18,400 18,400 G267-F423 267-GGHDAKF # ZnCl₂ and those of IA, IB, IIIA,and IVC were determined in samples with no ZnCl₂. Calculated masses arefor the sequence ranges shown based on N-terminal sequence and massdata. The N-terminal sequences were determined separately for IA, IB,and IIIA in solutions and for IIIB, IVA, IVB and IVC on PVDF membraneafter separation by SDS-PAGE and transfer on membrane. # IIIA as havinga sequence of V1-F266 with a mass of 32,270 was based on N-terminalamino acid sequence determination and SDS-PAGE results (FIGS. 10 and11).

Example 19 Other Analytical Methods

[0115] The enzymatic assay was based on HPLC separation and measurementof the nicked products from a 17-residue C-terminal peptide of SNAP-25corresponding to residues 187-203 (Schmidt and Bostian, 1995). Initiallyprotein concentrations were determined by BCA assay (Pierce) with bovineserum albumin (BSA) as a standard. After it was established by repeatedmeasurements that a 1 -mg/ml BoNT/A LC thus determined has A^(0.1%) (1cm light path) value of 1.0 at 278 nm (0.98 at 280 nm), proteinconcentration was determined from absorbance at 278 nm. For comparison,the calculated A^(0.1%) value of the LC at 280 nm in water (Pace et al.,1995) is 0.948. Absorption spectra were recorded in a Hewlett-Packard8452 diode array spectrophotometer. The N-terminal amino acid sequenceof the LC was determined by Edman degradation in the Applied BiosystemsProcise Sequences in the 0- to 20-pmol detection range.

Example 20 A Specific Competitive Inhibitor of LC Activity Was anEffective Inhibitor of Truncation and Fragmentation

[0116] Autocatalytic truncation and fragmentation of proteins can arisefrom chemical catalysis and from enzymatic catalysis. To differentiatethese two possibilities, a peptide specifically synthesized as acompetitive inhibitor of BoNT/A proteolytic activity (Schmidt et al.,1998) was used. This peptide inhibitor, with a sequence of CRATKML (SEQID. NO:46), competitively inhibits the cleavage of a 17-residuesubstrate peptide based on SNAP-25 by BoNT/A neurotoxin with a K_(i) of2 uM (Schmidt et al., 1998). At a 1 mM inhibitor peptide concentration,the LC showed no activity when assayed for 5 min. FIG. 12B shows thatwhen the LC was incubated with 1 mM peptide inhibitor, both C-terminaltruncation and fragmentation at the interior of LC were largelyprevented. In the presence of the peptide inhibitor, however, the LCunderwent a very slow cleavage, as can be expected in an enzymaticactivity with a competitive inhibitor. Densitometric scanning of the gelshowed that after 28 days, in the presence of the peptide inhibitor,less than 10% of the LC (IA) was converted into the C-terminallytruncated form (IB). In contrast, in the absence of the peptideinhibitor, more than 80% of the LC (IA) was converted into the truncatedform (IB). Results of this experiment prove that loss of 10-28 residuesfrom the C-terminus of LC followed by fragmentation into two majorpeptides (FIGS. 10 and 11, Tables 4 and 5) occurred at the active siteof the LC and that these reactions were enzymatic. The results alsoprovide direct evidence that the cleavage reactions were not due to anycontaminating protease in the preparation of the LC.

Example 21 Materials

[0117] PCR-TOPO and 1-Shot cells were from Invitrogen. pET24a plasmidand BL21 (DE3) cells were obtained from Novagen. All were prepared bystandard methods. Proteins were visualized by SDS-PAGE and stainedeither with Coomasie or Colloidal Coomasie (Novex). Westerns (Novex)were reacted with a rabbit primary antibody (Research Genetics, Inc.,Huntsville, Ala.) against the n-terminal 16 amino acids(PFVNKQFNYKDPVNGV) of the LC of type A and were visualized with ahorseradish peroxidase conjugated goat anti-rabbit secondary anti-bodyand TMB peroxidase substrate (Kirkegaard and Perry Laboratories).Bacterial media was from Difco. Purification of the expressed proteinswas on a Pharmacia model 500 FPLC system with programmed elution andA₂₈₀ monitoring (Pharmacia, Uppsala, Sweden). Columns were a PharmaciaHR 10/10 Mono S cation-exchange column, a Pharmacia Mono S 5/5 cationexchange column, and a Perseptive Biosystems POROS 20 HS cation exchangecolumn. Pretreatment of the expressed proteins was with DNAse (Sigma,Inc.) and dialysis was with Pierce Slide-A-Lyzer 10 k MWCO cassettes.The SNAP-25 substrate peptide (Quality Controlled Biochemicals,Hopkinton, Mass.) and its cleavage products were separated on a Hi-PoreC18 column, 0.45×25 cm (Bio-Rad Laboratories) and analyzed with theMillennium Software Package (Waters, Inc.). Src (p60c-src) recombinantphosphokinase, substrate peptide, and anti-phosphotyrosine monoclonalantibody 4G10 were from Upstate Biotechnology, Lake Placid, N.Y.[γ-³²P]ATP, 3000 Ci/mmol, was from Dupont-NEN.

Example 22 Preparation of Recombinant Neurotoxin Clones

[0118] New restriction sites were added by PCR to the 5′ and 3′ ends(Ndel and HindIII, respectively) of the synthetic DNA molecules codingfor the Lc (M₁, to K₄₄₉), the Lc plus belt (LC+Belt; M₁, to F₅₅₀) andthe Lc plus translocation region (LC+Xloc; M₁ to Q₆₅₉). These sequencescorrespond to GenBank accession numbers x, y and z respectively. PCRproducts were subcloned into pCR-TOPO and the sequences confirmed by DNAsequencing. The inserts were cut from the subcloning vector and ligatedbehind the Ndel site of pET24a, so as to begin expression with theinitial methionine of the LC. The plasmid was transformed into E. coliBL21 (DE3) cells for expression.

Example 23 Expression of Neurotoxins

[0119] One hundred ml of Terrific Broth (TB) plus kanamycin wasinoculated with the appropriate clone and grown overnight, with shaking,at 37° C. Fifty ml of LcA or 100 ml LcA+Belt and Lc+Hn of overnightgrowth was added to 1 liter TB plus kanamycin and shaking incubationcontinued at 37° C. for an additional 1.25 hours. While cultures wereplaced on ice for 5 to 10 minutes, the OD₆₀₀ was read and adjusted toapproximately 0.4 to 0.6, then IPTG was added to 1 mM for induction ofprotein expression. Duplicate cultures were grown at 37° C. (4 hours),30° C. (10 hours) and 18° C. (22 hours). At harvesting, the OD₆₀₀ wasread again, cells were pelleted and frozen at −70° C. if not usedimmediately. Data points are the mean of three separate measurements ofthe appropriate bands from SDS-PAGE gels scanned and digitally analyzedwith an AlphaImager 2000 densitometer and AlphaImager Documentation andAnalysis Software (AlphaInotech, San Leandro, Calif.).

Expression at Low Temperatures Markedly Increases Yields of SolubleProduct, While Addition of Portions of the Hn Does Not Increase theYield of Soluble Product

[0120] To study the effects of low temperature induction on theexpression of LcA, expression was performed at 18° C., 30° C. and 37° C.FIG. 1 5A shows the decreasing solubility of LcA at these threetemperatures, with concomitant decrease in the soluble product, from55.5% at 18° C. to 5.2% at 37° C. Yields of soluble LcA were highest at18° C., with LcA making up approximately 10% of the cell protein.Addition of the belt and Hn portions of the neurotoxin to LcA did notincrease solubility (FIGS. 15A, 15B and 15C), although addition of thefull Hn region reduced expression and yield (FIG. 15C).

[0121] Constructs were grown both in Luria Broth (LB) and Terrific Broth(TB), with no apparent difference in the quality or percent solubilityof the products. Total yield was far greater for growth in TB, 17.97 g/lverses 7.77 g/l for LB. Optimal expression conditions for the Lc wereconsidered to be the construct lacking either the belt or the Hn regionat 18° C. for 20-24 hours in TB.

Example 24 Sample Preparation and Purfication of LC

[0122] One gram E. coli cell paste was resuspended into 20 ml of bufferA (20 mM NaAcetate, 2 mM EDTA, pH5.4). The suspended cells weredisrupted by sonicating for 12 cycles of 30 seconds followed by 30seconds of incubation on ice using a medium size probe at 65% output.The resulting cell lysate was centrifuged (Sorval) at 15,000×g for 15minutes at 4° C. to separate the proteins into soluble and insolublefractions. The soluble fraction was diluted 1:1 in equilibration bufferB (20 mM NaAcetate, 2 mM EDTA, pH5.8) and used as starting material forthe chromatography.

[0123] A HR 10/10 Mono S cation-exchange column was extensively cleanedbetween runs by sequentially running through it: 1 M NaCl through at 3ml/min for 5 minutes; 20 mM NaOH for 10 minutes at 1 ml/min; 70% ethanolin ddwater for 30 minutes at 1 ml/min; 1 M NaCl in buffer B for 15minutes at 1 ml/min; then re-equilibrated with buffer B at 2 ml/min for5 minutes. The diluted lysate was then loaded at a flow rate of 2 ml/min(150 cm/h). The column was washed with 24 ml (3 bed volumes) of bufferB. Flow through and wash were collected separately and stored forsubsequent analysis. Protein was eluted from the column with a lineargradient from 0 to 70% 1 M NaCl in buffer B over 8 minutes. Two-mlfractions were collected throughout the gradient. Fractions elutingbetween 10 and 22 mSiemanns (mS) were positive for rBoNTA(Lc) as shownby Western blot analysis. The pooled fractions were diluted 1:3 withbuffer C (20 mM NaAcetate, 2 mM EDTA, pH6.2) and loaded onto a Mono S5/5 cation exchange column equilibrated with buffer C at a flow rate of2.5 ml/min. The column was washed with 10 ml (10 bed volume) of bufferC. Protein was eluted from the column with a linear gradient of 0-75% 1M NaCl in buffer C over 15 minutes. The rBoNTA(Lc) protein eluted fromthe Mono S column as a single band at 12 mS as shown by Western blotanalysis. Fractions were pooled and stored frozen at −20° C. in plasticvials. The product was greater than 98% pure as determined by SDS-PAGE.

[0124] The LcA+belt and the LcA+Hn were similarly purified, except thatsonication was in buffer A (20 mM NaAcetate, 2 mM EDTA buffer, pH 4.8)and dilution was not necessary after centifugation to obtain the solublefraction. After extensive cleaning of the column, the soluble fractionsof either LcA+Belt or LcA+Hn were loaded at 2 ml/min onto a Poros 20 HScolumn equilibrated with buffer A. After loading, the column was rinsedat 3 ml/min with buffer A for 5 minutes and a 5% step of 1 M NaCl inbuffer A was performed to remove interferring cellular products. TheLcA+Belt was then eluted with a 9% step and the LcA+Hn eluted with a10-14% step of 1 M NaCl in buffer A. Fractions were pooled, diluted 1:3with equilibration buffer A and re-run on the HS column, eluting with a1 to 75% gradient of 1 M NaCl in buffer A. Verification of the peaks wasby Western blot and SDS-PAGE. Each protein was 95% or greater pure.

[0125] Fractions were pooled and stored frozen at −20° C. in plasticvials.

[0126] After the first column purification, aliquots of the expressedLcA+Hn were additionally nicked with trypsin at 10 □g/ml overnight, atroom temperature. This semi-purified protein lysate was then diluted andrun on a second Poros HS column as described above. Protein wassimilarly 95% or greater pure.

[0127] Total protein concentrations were determined by using either aBio-Rad Protein assay at one-half volume of the standard protocol andbovine serum albumen as the protein standard or the Pierce BCA(bicinchoninic acid) protein assay with the microscale protocol asdirected, with bovine serum alubumin as the protein standard.

Purification of the Lc From the Soluble Fraction of the LowestTemperature Expressed

[0128] Once conditions had been achieved for optimal yield of product,recovery of the Lc by simple cell sonication was deemed sufficient torelease the protein. After removal of insoluble cell debris and proteinsby centrifugation, this extract was directly loaded onto a cationexchange column and two isoforms of the Lc were observed to elutebetween 180 and 280 mM NaCl (FIG. 16A). Western analysis of collectedfractions showed two peaks reactive to antisera, corresponding to a fulllength Lc, and a Lc truncated by approximately 2.5 kDa. Since both formswere reactive to the amino terminus specific sera, a carboxy terminustruncation was indicated. The calculated pI for a Lc lacking theterminal 21 residues is 6.39, suggesting that it would be eluted at alower NaCl concentration, as was observed. This difference in elutionconditions allowed for a separate purification of each Lc isoform. Theproducts eluted from the cation exchange chromatography column wereobserved to be approximately 70% pure, with a total proteinconcentration of 1.1 mg/ml.

[0129] The material was reloaded onto the Mono S column for furtherpurification. The larger, non-truncated, LcA eluted as a single peak at12 mS (FIG. 16B). SDS-Page and western blot analysis showed only asingle band at 51 k-Da (FIGS. 17A and 17B). The product was judged to be98% pure after the final step and a protein determination determined theoverall yield was 0.53 mg purified Lc per gram wet cells obtainable fromour protocol.

[0130] The LcA+Belt eluted from the first column purification wasapproximately 85% pure, with a protein concentration of 0.454 mg/ml, ina total of 12 ml (FIG. 2C). After purification on the second column, a 4ml pooled peak (FIG. 16D) had a concentration of 0.226 mg/ml, with 98%purity, producing a single band as observed by Western analysis (FIG.17A and 17C). The overall yield was 0.347 mg/gm wet cells.

[0131] The LcA+Hn eluted from the first column purification wasapproximately 80% pure, with a protein concentration of 0.816 mg/ml, ina total of 12 ml (FIG. 6D). After purification on the second column, a 4ml pooled peak (FIG. 16E) had a concentration of 0.401 mg/ml, with 98%purity, forming a single band, while the nicked form of the constructproduced two bands (FIGS. 17A through 17D) corresponding to the Hn andLc. The overall yield was 0.617 mg/gm wet cells. The nicked form of theconstruct

Example 25 Assay for Cleavage of SNAP-25 Peptide

[0132] A 17-residue C-terminal peptide of SNAP-25(acetyl-SNKTRIDEANQRATKML-amide) shown to be the minimum length requiredfor optimal BoNt/A proteolytic activity (Schmidt and Bostian, 1997) wasused as the substrate in a cleavage assay as described previously(Ashraf et al, in press). Briefly, a 0.3 ml mixture containing 0.7-1.0mM of the substrate peptide, 0.25 mM ZnCl₂, 5.0 mM DTT, 50 mM Na-HEPESbuffer (pH=7.4) and purified LC (adjusted to produce 10-30% finalcleavage) was incubated at 37° C. for 15-180 minutes. The reaction wasstopped with 0.09 ml of 0.7% trifluoroacetic acid. Quantitation ofcleaved and uncleaved peptide was done by reverse-phase HPLC separationand the fraction of the peptide proteolysed was calculated by dividingthe combined areas of the two cleaved peaks by the sum of the twoproduct and uncleaved substrate peaks.

Catalytic Activity of the Expressed Constucts

[0133] Incubation of the 17-mer synthetic peptide representing residues187-203 of SNAP-25 with the purified Lc at 37° C. generates only twopeptides cleaving between residues 197 (glutamine) and 198 (arginine).No other peptide fragments were generated by prolonged incubation,indicating that any contaminants in the Lc preparation lackedproteolytic activity. FPLC purification run #71, which was the completeLc, resulted in a specific activity of 2.36 μmol/min/mg of Lc. NativeBoNT/A in previous assays with the SNAP-25 synthetic peptide had aspecific activity of 0.241 μmol/min/mg (Schmidt and Bostian). Thus, thepurified Lc produced had a specific activity increased by approximately10-fold. Run #32 was the Lc+belt, and had an activity of 0.08μmol/min/mg.

Example 26 Determination of the Length of the Purified Whole andTruncated Lc

[0134] HPLC-purified samples were mixed with sinapinic acid anddeposited on a stainless steel target. Mass spectra were acquired with aPerseptive Biosystems Voyager DE MALDI-TOF system. Data were obtained indelayed extration mode (750 ns delay) with a 337 nm nitrogen laser (3 nswide pulse), using an acquisition rate of 2 GHz, 50,000 channels, anaccelerating voltage of 25000, 93% grid voltage, and a 0.3% guide wirevoltage. Typically, 128 scans were averaged. The mass spectrometer wasexternally calibrated with myoglobin and bovine serum albumin.

[0135] The amino-terminal sequence of the expressed Lc was determined byautomated Edman degradation performed on an Applied Biosystems ProciseSequencer (Applied Biosystems, Foster City, Calif.) in the 0-20 picomoledetection range.

Determination of the Cleavage Point for Purified Lc

[0136] Purified Lc kept at −20° C. in purification buffer with 2 mM EDTAhad no observable cleavage or truncation products (FIG. 18A, lane 1).When the same product was placed at 30° C. for 1 hour, the truncated Lcseen after the first cation exchange column passage was observed (FIG.18A, lane 2). FIG. 18 contains the mass spectrum for cleaved BoNT/A Lc.The ion at mlz 49039.0 corresponds to the singly-charged molecule,whereas ions at m/z 24,556.9, and 98,280 correspond to doubly-chargedand dimer species, respectively. The verified amino terminus for the Lcwas VQFVNKQFNY, with the terminal methione removed, resulting in apeptide of 448 residues. The observed principal mass of 49,039 isapproximately 2279 daltons less than the calculated mass for type A Lc,which represents a loss of 21-22 amino acids. Since the amino terminusspecific antibody still reacts with the truncated molecule, cleavageoccurred near the C terminus of the molecule. Because of massuncertainty with MALDI-TOFMS (0.05% maximum mass accuracy for thisinstrument), it was not possible to positively identify the site ofcleavage. Nevertheless, it was determined that cleavage occurred ateither Y₄₂₆, K₄₂₇, or L₄₂₈. The most probable site of cleavage wasbetween K₄₂₇ and L₄₂₈. Calculated mass for that product was 48,999, adifference of 40 daltons, which represents the best match to theobserved ion and a mass accuracy to within 0.08%.

[0137] Addition of MgCl₂ to 125 mM and incubation for 1 hour at 30° C.resulted in two cleavage products (FIG. 18A, lane 3) after the Lc hadlost the carboxy terminal residues. Amino terminus sequencing showed thecleavage to be between two tyrosines, Y₂₅₀ and Y₂₅₁.

Example 27 Phosphorylation of Purified Lc

[0138] Phosphorylation was at 30° C. for 1 to 24 hours in a finalreaction volume of 40 μL with 30 units c-src kinase. Non-phosphorylatedsamples were those in which enzyme b was omitted. The amount of Lc inthe reaction was from 6.25 nM to 1.25 nM. The 4×buffer used for thereaction consisted of 100 mM Tris-HCI, pH 7.2, 125 mM MgClz, 25 mMMnCl2, 2 mM EGTA, and 2 mM DTT. ATP was at either 500 μM or 1 mM, with[γ-³²P]ATP added to a final concentration of 1 μCi/ul. In some cases,substrate peptide (KVEKIGEGTGVVYK; SEQ ID NO:3) at 93 μM was substitutedfor the Lc to act as a control. Reactions were stopped by freezing at−20° C. Phosphorylated samples were run on SDS-PAGE gels, and eitherblotted and bands visualized with an antibody specific to phosphorylatedtyrosine or the amino terminus of the Lc, or they were stained withCoomasie Blue, destained, dried and exposed to Kodak BioMax Light film.

Phosphorylation of Lc

[0139] Purified Lc that was tyrosine phosphorylated resisted cleavage atthe Y₂₅₀-Y₂₅₁ site (FIG. 18B). During the initial 1 hour period ofphosphorylation, the characteristic cleavage products were observed, butdid not substantially increase over a 24 hour period of time. A possibleexplanation would be that phosphorylated Lc molecules were protectedfrom cleavage, but not all of them could be modified prior to concurrentproteolysis. An identical reaction mixture lacking the enzyme showedrapid cleavage of the Lc, with very little remaining by 4 hours, andundetectable by 8 hours. A monoclonal antibody to phosphorylatedtyrosine reacted to full length, src phorphorylated Lc, but not toeither of the cleavage products in the phosphorylation reaction (FIG.19B), even though cleavage products were clearly visible by SDS-PAGE atall time points. The reaction lacking the enzyme showed nophosphorylated tyrosine bands of any size. Antibody to the aminoterminus of the Lc reacted to the full length and larger of the cleavageproducts, plus three additional bands of between 60 and 75 kDa. Theseadditional bands above the Lc were observed by SDS-PAGE for all thesamples and appear to be SDS-resistant complexes of either the Lc oramino terminus fragment with other fragments. Autoradiographs of thephosphorylated and unphosphorylated (lacking enzyme) Lc (FIG. 19C) showincorporation of [γ-³²P]ATP in the src phosphorylated full length Lc at1 hour, with none observed in smaller or larger fragments, nor insamples lacking the enzyme. At 24 hours, very faint bands correspondingto the cleavage products did appear. These could either have arisen fromcleaved, phosphorylated, full length Lc, or they may have beenphosphorylated after they became fragments.

Example 28 Immunity

[0140] Immunization of mice with the purified forms of the LcA, LcA+beltand LcA+Hn resulted in ELISA titers of between X and X for all constructforms. Protection was observed after challenge with 10² to 10³ MLD₅₀ ofpurified Type A toxin. See Tables 6-8. TABLE 6 Efficacy of PurifiedrBoNTA(LC + Belt) Solubly Expressed from E. coli to Elicit ProtectiveImmunity in Mice Toxin Challenge Dosage^(a,b) (Survivors/Total) ELISATiter (μg) 10²LD₅₀ 10³LD₅₀ (GMT)^(c)  5 10/10 10/10 ND 15 10/10 10/10 NDControls  0/10  0/10 ND

[0141] TABLE 7 Efficacy of Purified rBoNTA(LC + Hn) Solubly Expressedfrom E. coli to Elicit Protective Immunity in Mice Toxin ChallengeDosage^(a,b) (Survivors/Total) ELISA Titer (μg) 10²LD₅₀ 10³LD₅₀(GMT)^(c)  5 5/9  1/9  ND 15 4/10 1/10 ND Controls 0/10 0/10 ND

[0142] TABLE 8 Efficacy of Purified rBoNTA(LC) Solubly Expressed from E.coli to Elicit Protective Immunity in Mice Toxin Challenge Dosage^(a,b)(Survivors/Total) ELISA Titer (μg) 10²LD₅₀ 10³LD₅₀ (GMT)^(c)  5 9/1010/10 ND 15 9/10 10/10 ND Controls 0/10  0/10 ND

Example 29 Discussion

[0143] The system of expression of the invention for botulinumneurotoxin Hc (Byrne et al, 1998) and Lc (Ahmed et al, in prep.)fragments using an optimized synthetic gene, has previously shownsuccess in achieving high levels of product. In an attempt to produce amolecule that more closely resembles the natural state of the toxin, acloning and expression scheme that would give a large amount ofcorrectly folded, untagged, Lc was initiated. The two basic strategiesemployed were to (1) express the Lc at a lower temperature, a classicmethod for ensuring proper folding, and (2) adding on portions of therest of the neurotoxin polypeptide, mimicking the natural expressionwithin the clostridial host. As expected, reducing the temperature forinduction dramatically increased the solubility of the expressed productfrom 5.2% at 37° C. to 55.5% at 18° C. for the Lc. The slower rate ofexpression at the lower temperatures was compensated for by increasingthe length of time for expression. This did not result in increaseddegradation of the product intracellularly, prior to harvest andpurification. Addition of the belt and Hn portions of the toxin had noeffect upon solubility of the expressed gene, although each was easilyexpressed at the lower temperature.

[0144] Although cloned and expressed Lc has been available for Lc study,it has been purified with either glutathione or his-tags (Zhou, et al,1995; Li and Singh, 1999). Previous investigators have used native toxin(Lacy et al, 1998) for x-ray crytallography studies, and it was anobject of the invention to produce Lc as close to the native product aspossible, e.g., without tags or modifications. For this reason,traditional column chromatography methods were used instead of affinitycolumns. The calculated pI of the Lc of 8.13 suggested that the Lc wouldefficientlt bind to a cation exchange column. Upon passage over aninitial Mono S column, the product appeared relatively clean, although asecond immunoreactive band immediately beneath the proper, calculatedsize for the Lc was noted. After passage over a second cationic exchangecolumn, this band was not observed on Westerns.

[0145] Using the above methods of low temperature expression and cationexchange purification, a large quantity of Lc was acquired forassessment of catalytic activity. Activity of the purified Lc wascalculated to be approximately 10-fold greater than that of the nativetoxin. Previous investigators have shown that the Lc must be activatedby proteolytic cleavage of the Lc from the Hc (DasGupta and Dekleva,1990), although the two halves must both be present for efficientintoxication of cells. It is interesting that the Lc with the beltattached lacked the high level of catalytic activity seen with the Lc byitself. Presumably, the belt is wrapped around the Lc, as is observed inx-ray crystallography studies (Lacy et al, 1998). As the entiretranslocation region is not there to occlude the active site, it may bethat the belt in some manner is constricting the Lc, or a conformationalchange is prevented that is required for full activation. Comparison ofthe crystallography structure of Lc of the invention with and withoutthe belt would be worth further study.

[0146] Two interesting and unexpected pieces of data came fromexpression of Lc without purification tags. The first was the truncationof the Lc from the carboxy terminus by 20 residues. A recent paper byKadkhodayan et al, 2000, notes that this portion of the Lc is notrequired for full catalytic activity. The truncation is intriguing as itremoves the Lc/Hc di-sulfide bond at a lysine proximal to the involvedcysteine. The two other proteolytic cleavages known to occur at thecarboxy teminus of the Lc are also at lysine residues (DasGupta andDekleva, 1990). Lysine proteolysis is common, with ubiquitin, a lysinespecific proteolysis factor found conjugated to cell receptors ofeukaryotes being one of the most common routes (Doherty and Mayer,1992). It has long been hypothesized that the di-sulfide bond holdingthe Lc and Hc together was reduced as the Lc was transported into thecell, freeing it from the receptor binding portion (de Paiva et al,1993). Although the ten residue portion flanked by lysine residues seemsto be removed during activation “nicking” of the polypeptide, thecysteine residue was assumed to remain as part of the Lc. Work withnative toxin and cells has been inititated to determine if the naturalstate of the toxin inside cells is one lacking the terminal 20 residuesand cysteine.

Example 30 Expression of BoNT LC

[0147] Reagents: Terrific Broth (Difco): 48 gm/liter with 4 ml ofnon-animal glycerol; autoclave 15 minutes. Store refrigerated.Kanamycin: stock solution is 50 mg/ml in distilled water, filtersterilized, store in aliquots at −20° C. Chloramphenicol: stock solutionis 50 mg/ml in ethanol, filter sterilized, store in aliquots at −20° C.Add antibiotics to media just prior to use.

[0148] Expression of the Lc and Lc with Hc (translocation region) wasperformed for even numbered SEQ ID NOS:20-44. Expression was essentiallythe same for all constructs within the given parameters.

[0149] Cultures of BL21(DE3) cells were grown in Terrific Broth (TB)plus 50 μg/mL kanamycin. Cultures of BL21(DE3) Codon Plus cells weregrown in TB plus 50 μg/mL kanamycin and 50 μg/mL chloramphenicol.Cultures grown overnight at 37° C. while shaking at about 200 to about250 rpm were diluted 1:20 with fresh antibiotic-containing media.Diluted cultures were returned to overnight growth conditions (37° C.,shaking at 200-250 rpm) for 1¼ to 2½ hours. An optical densitymeasurement was taken while the cultures were placed on ice for 5minutes. Preferably, the OD₆₀₀ is between about 0.4 and about 0.6. Theincubation time may be extended and/or fresh antibiotic-containing mediamay be added if the OD₆₀₀ is lower than 0.4 or higher than 0.6.

[0150] Next, sufficient IPTG was added to each chilled culture to makethe concentration about 1 mM. IPTG-containing cultures were incubatedabout 24 to about 26 hours at 18° C and shaking at about 200 to about250 rpm. An optical density measurement was taken at the end of thisincubation. Preferably, the OD₆₀₀ is between about 1.7 and about 2.1.

[0151] Cultures that satisfied this criteria were centrifuged at about3000 rpm for about 20 minutes to obtain a cell paste for purification.The cell paste may be stored at −20° C. until ready for use.

[0152] Aliquots of 1 mL each were pelleted in a microfuge, resuspendedin 1 mL of sonication buffer, and sonicated 12×30 seconds on ice over 12minutes. Sonicated cells were microfuged for 10 minutes. The supernatantwas aspirated and retained as the soluable fraction. 1 mL of 6M urea wasadded to each pellet and retained as the insoluable fraction.Appropriate amounts run on by SDS-PAGE should show approximately 50%soluble, 50% insoluble, at about 51 kDa. A western with rabbit anti-Lcsera will be at the same location.

Purification of BoNT LC

[0153] Cell paste was resuspended at 1 g/20 mL sonication buffer,sonicated 10X, 30 seconds on, 30 seconds off, on ice. Insoluablematerial and debris was pelleted by centrifuging for 10 minutes at12,000 rpm (e.g. in a microfuge), decanting solute, and repeating onetime in a fresh tube. The supernatant was decanted into a fresh tube. Anequal volume of equilibration buffer may be optionally added to thesupernatant to facilitate cation exchange chromatography, e.g. flow. Forexample, such dilution facilitates column loading and washing when usinga Source S resin from Pharmacia whereas such dilution is unnecessarywhen using a Poros cationic resin. Filter sterilize the supernatant with0.45 μm filters.

[0154] Run #1: A column (100 mm) was equilibrated with equilibrationbuffer, 2 minutes, 2.5 to 3 ml/min (same rate through out run). Cellpaste (20-40 mL per run) was manually loaded. The column was washed for3 minutes with equilibration buffer. Using gradient buffer, a 0 to 70%gradient was run over 8 minutes. For some cell lysates, a 5% NaCl (5 mS)5 minutes step was performed. For example, where a Source S resin wasused, no salt wash was was performed, but where a Poros resin was used,this salt wash was performed to elute contaminating proteins. Cellprotein was collected at between 10 and 22 mS. Fractions (1 mL) werecollected through out the gradient. The desired protein will elute atbetween 10 and 22 mS, depending upon the expression product used.

[0155] Run #2: The peak fractions from run #1 were pooled. Equilibrationbuffer was added to pooled fractions, at a 3:1 ratio. The column wasequilibrated with equilibration buffer for 2 minutes, at 2.5 to 3 ml/min(same rate through out run). The run #1 pool was loaded onto the column;washed 2 minutes with equilibration buffer. Using gradient buffer, a 0to 75% gradient was run over 15 minutes. Fractions (1 mL) were collectedand peak fractions were pooled. Aliquots of the pooled fractions werestored in plastic vials at −20° C.

[0156] A portion of the purified protein was used to measure theA_(260/278). The ratio may be used as a measure of the presence of DNAand the A₂₈₀ to quantitate the protein by using the calculated molarextinction coefficient and molecular weight.

[0157] A cleaning procedure must be done on the column between each run.Run 1 M NaCl through column at 3 ml/min for 5 minutes. Run 20 mM NaOHthrough the column at 1 ml/min for 10 minutes. Run 70% ETOH through thecolumn at 1 ml/min for 30 minutes. Run 1 M NaCl through it at 1 ml/minfor 15 minutes. Re-equilibrate the column to the proper pH with a lowsalt buffer.

Buffers

[0158] A combination of sonication buffers, equilibration buffers andgradient buffers is used for each cell lysate. Sonication buffers arealways chosen to be 0.4 pH below the equilibration buffer. Gradientbuffers are the same as equilibration buffers except for addition of 1 MNaCl.

[0159] Gradient buffer A: 55 mM Na mono-phosphate, 2 mM EDTA, 1 M NaCl,in milliQ water; pH to 5.8; filter. Gradient buffer B: 20 mM NaAcetate,1 M NaCl, in milliQ water, pH to 5.4, filter. Gradient buffer C1: 20 mMNaAcetate, 1 M NaCl, in milliQ water, pH to 4.8, filter. Gradient bufferC2: 20 mM NaAcetate, 2 mM EDTA, 1 M NaCl, in milliQ water, pH to 5.4,filter. Gradient buffer D: 20 mM NaAcetate, 2 mM EDTA, 1 M NaCl, inmilliQ water, pH to 4.8, filter.

Results

[0160] Expression and purification of BoNT/A LC according to this methodyielded protein with a specific activity (SNAP-25 assay) that was about10-fold higher than when BoNT/A LC was purified from inclusion bodies(Ahmed and Smith (2000) J. Prot Chem. 19, 475-487).

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1 47 1 16 PRT Clostridium botulinum PEPTIDE (0)...(0) N-terminalresidues of mature, wild-type botulinum neurotoxin 1 Pro Phe Val Asn LysGln Phe Asn Tyr Lys Asp Pro Val Asn Gly Val 1 5 10 15 2 17 PRT HumanPEPTIDE (0)...(0) Residues 187-203 of SNAP-25 2 Ser Asn Lys Thr Arg IleAsp Glu Ala Asn Gln Arg Ala Thr Lys Met 1 5 10 15 Leu 3 14 PRTArtificial Sequence Synthetic peptide; control for phosphorylationexperiments 3 Lys Val Glu Lys Ile Gly Glu Gly Thr Gly Val Val Tyr Lys 15 10 4 1403 DNA Artificial Sequence Synthetic botulinum neurotoxin lightchain of serotype A based on wild-type Clostridium botulinum sequence 4gaattcccat ggttcagttc gttaacaaac agttcaacta caaagacccg gttaacggtg 60ttgacatcgc ttacatcaaa atcccgaacg ttggtcagat gcagccggtt aaagcattca 120aaatccacaa caaaatctgg gttatcccgg aacgtgacac tttcactaac ccggaagaag 180gtgacctgaa cccgccgccg gaagctaaac aggttccggt ttcttactac gactctactt 240acctgtctac tgacaacgaa aaggacaact acctgaaagg tgttactaaa ctgtttgaac 300gtatctactc tactgacctg ggtcgcatgc tgctcacttc tatcgttcgt ggtatcccgt 360tctggggtgg ttctactatc gacactgaac tgaaagttat cgacactaac tgcatcaacg 420ttatccagcc ggacggttct taccgttctg aagaactgaa cctggttatc atcggtccgt 480ctgctgacat catccagttt gaatgcaaat ctttcggtca cgaagttctg aacctgactc 540gtaacggtta cggttctact cagtacatcc gtttctctcc ggacttcact ttcggtttcg 600aagaatctct ggaagttgac actaacccgc tgctgggtgc tggtaaattc gctactgacc 660cggctgttac tctggctcac gaactgatcc acgctggtca ccgtctgtac ggtatcgcta 720tcaacccgaa ccgtgttttc aaagttaaca ctaacgctta ctacgaaatg tctggtctgg 780aagtttcttt tgaagaactg cgtactttcg gtggtcacga cgctaaattc atcgactctc 840tgcaggaaaa cgagttccgt ctgtactact acaacaaatt caaagacatc gcttctactc 900tgaacaaagc taaatctatc gttggtacca ctgcttctct gcagtacatg aagaacgttt 960tcaaagaaaa gtacctgctg tctgaagaca cttctggtaa attctctgtt gacaaactga 1020aattcgacaa actgtacaaa atgctgactg aaatctacac tgaagacaac ttcgttaaat 1080tcttcaaagt tctgaaccgt aaaacttacc tgaacttcga caaagctgtt ttcaaaatca 1140acatcgttcc gaaagttaac tacactatct acgacggttt caacctgcgt aacactaacc 1200tggctgctaa cttcaacggt cagaacactg aaatcaacaa catgaacttc actaaactga 1260agaacttcac tggtctgttt gagttctaca aactgctgtg cgttcgtggt atcatcactt 1320ctaaaactaa atctctggac aaaggttaca acaaactggt tccgcgtggt tctcatcatc 1380atcatcatca ttaatgagaa tcc 1403 5 461 PRT Artificial Sequence Syntheticbotulinum neurotoxin light chain of serotype A based on wild-typeClostridium botulinum sequence 5 Met Val Gln Phe Val Asn Lys Gln Phe AsnTyr Lys Asp Pro Val Asn 1 5 10 15 Gly Val Asp Ile Ala Tyr Ile Lys IlePro Asn Val Gly Gln Met Gln 20 25 30 Pro Val Lys Ala Phe Lys Ile His AsnLys Ile Trp Val Ile Pro Glu 35 40 45 Arg Asp Thr Phe Thr Asn Pro Glu GluGly Asp Leu Asn Pro Pro Pro 50 55 60 Glu Ala Lys Gln Val Pro Val Ser TyrTyr Asp Ser Thr Tyr Leu Ser 65 70 75 80 Thr Asp Asn Glu Lys Asp Asn TyrLeu Lys Gly Val Thr Lys Leu Phe 85 90 95 Glu Arg Ile Tyr Ser Thr Asp LeuGly Arg Met Leu Leu Thr Ser Ile 100 105 110 Val Arg Gly Ile Pro Phe TrpGly Gly Ser Thr Ile Asp Thr Glu Leu 115 120 125 Lys Val Ile Asp Thr AsnCys Ile Asn Val Ile Gln Pro Asp Gly Ser 130 135 140 Tyr Arg Ser Glu GluLeu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp 145 150 155 160 Ile Ile GlnPhe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu 165 170 175 Thr ArgAsn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp 180 185 190 PheThr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu 195 200 205Leu Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His 210 215220 Glu Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro 225230 235 240 Asn Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met SerGly 245 250 255 Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly HisAsp Ala 260 265 270 Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg LeuTyr Tyr Tyr 275 280 285 Asn Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn LysAla Lys Ser Ile 290 295 300 Val Gly Thr Thr Ala Ser Leu Gln Tyr Met LysAsn Val Phe Lys Glu 305 310 315 320 Lys Tyr Leu Leu Ser Glu Asp Thr SerGly Lys Phe Ser Val Asp Lys 325 330 335 Leu Lys Phe Asp Lys Leu Tyr LysMet Leu Thr Glu Ile Tyr Thr Glu 340 345 350 Asp Asn Phe Val Lys Phe PheLys Val Leu Asn Arg Lys Thr Tyr Leu 355 360 365 Asn Phe Asp Lys Ala ValPhe Lys Ile Asn Ile Val Pro Lys Val Asn 370 375 380 Tyr Thr Ile Tyr AspGly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala 385 390 395 400 Asn Phe AsnGly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys 405 410 415 Leu LysAsn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val 420 425 430 ArgGly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn 435 440 445Lys Leu Val Pro Arg Gly Ser His His His His His His 450 455 460 6 1323DNA Artificial Sequence Synthetic botulinum neurotoxin light chain ofserotype B based on wild-type Clostridium botulinum sequence 6atgccagtta ctattaacaa cttcaactac aacgacccaa ttgacaacaa caacattatt 60atgatggagc caccattcgc tagaggtact ggtagatact acaaggcttt caagattact 120gacagaattt ggattattcc agagagatac actttcggtt acaagccaga ggacttcaac 180aagtcttctg gtattttcaa cagagacgtt tgtgagtact acgacccaga ctacttgaac 240actaacgaca agaagaacat tttcttgcaa actatgatta agttgttcaa cagaattaag 300tctaagccat tgggtgagaa gttgttggag atgattatta acggtattcc atacttgggt 360gacagaagag ttccattgga ggagttcaac actaacattg cttctgttac tgttaacaag 420ttgatttcta acccaggtga ggttgagaga aagaagggta ttttcgctaa cttgattatt 480ttcggtccag gtccagtttt gaacgagaac gagactattg acattggtat tcaaaaccac 540ttcgcttcta gagagggttt cggtggtatt atgcaaatga agttctgtcc agagtacgtt 600tctgttttca acaacgttca agagaacaag ggtgcttcta ttttcaacag aagaggttac 660ttctctgacc cagctttgat tttgatgcac gagttgattc acgttttgca cggtttgtac 720ggtattaagg ttgacgactt gccaattgtt ccaaacgaga agaagttctt catgcaatct 780actgacgcta ttcaagctga ggagttgtac actttcggtg gtcaagaccc atctattatt 840actccatcta ctgacaagtc tatttacgac aaggttttgc aaaacttcag aggtattgtt 900gacagattga acaaggtttt ggtttgtatt tctgacccaa acattaacat taacatttac 960aagaacaagt tcaaggacaa gtacaagttc gttgaggact ctgagggtaa gtactctatt 1020gacgttgagt ctttcgacaa gttgtacaag tctttgatgt tcggtttcac tgagactaac 1080attgctgaga actacaagat taagactaga gcttcttact tctctgactc tttgccacca 1140gttaagatta agaacttgtt ggacaacgag atttacacta ttgaggaggg tttcaacatt 1200tctgacaagg acatggagaa ggagtacaga ggtcaaaaca aggctattaa caagcaagct 1260tacgaggaga tttctaagga gcacttggct gtttacaaga ttcaaatgtg taagtctgtt 1320aag 1323 7 441 PRT Artificial Sequence Synthetic botulinum neurotoxinlight chain of serotype B based on wild-type Clostridium botulinumsequence 7 Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile AspAsn 1 5 10 15 Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly ThrGly Arg 20 25 30 Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile IlePro Glu 35 40 45 Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys SerSer Gly 50 55 60 Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp TyrLeu Asn 65 70 75 80 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met IleLys Leu Phe 85 90 95 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu LeuGlu Met Ile 100 105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg ValPro Leu Glu Glu 115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val AsnLys Leu Ile Ser Asn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly IlePhe Ala Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu AsnGlu Asn Glu Thr Ile Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala SerArg Glu Gly Phe Gly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro GluTyr Val Ser Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala SerIle Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile LeuMet His Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly IleLys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 PheMet Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280285 Tyr Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290295 300 Lys Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr305 310 315 320 Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp SerGlu Gly 325 330 335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu TyrLys Ser Leu 340 345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu AsnTyr Lys Ile Lys 355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu ProPro Val Lys Ile Lys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr IleGlu Glu Gly Phe Asn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys GluTyr Arg Gly Gln Asn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu GluIle Ser Lys Glu His Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys LysSer Val Lys 435 440 8 1332 DNA Artificial Sequence Synthetic botulinumneurotoxin light chain of serotype C1 based on wild-type Clostridiumbotulinum sequence 8 atgccaatca ccatcaacaa cttcaactac tcagaccctgtcgacaacaa gaacattctg 60 tacctggaca ctcacctgaa caccctagct aacgagcctgagaaggcctt tcggatcacc 120 ggaaacatct gggtcatccc tgatcgtttc tcccgtaactccaaccccaa cctgaacaag 180 cctcctcggg tcaccagccc taagagtggt tactacgaccctaactacct gagtaccgac 240 tctgacaagg acaccttcct gaaggagatc atcaagctgttcaagcgtat caactcccgt 300 gagatcggag aggagctcat ctacagactt tcgaccgatatccccttccc tggtaacaac 360 aatactccaa tcaacacctt cgacttcgac gtcgacttcaactccgtcga cgtcaagact 420 cggcagggta acaactgggt taagactggt agcatcaacccttccgtcat catcactgga 480 cctcgtgaga acatcatcga cccagagact tccacgttcaagctgactaa caacaccttc 540 gcggctcaag aaggattcgg tgctctgtca atcatctccatctcacctcg tttcatgctg 600 acctactcga acgcaaccaa cgacgtcgga gagggtaggttctctaagtc tgagttctgc 660 atggacccaa tcctgatcct gatgcatgag ctgaaccatgcaatgcacaa cctgtacgga 720 atcgctatcc caaacgacca gaccatctcc tccgtgacctccaacatctt ctactcccag 780 tacaacgtga agctggagta cgcagagatc tacgctttcggaggtccaac tatcgacctt 840 atccctaagt ccgctaggaa gtacttcgag gagaaggctttggattacta cagatccatc 900 gctaagagac tgaacagtat caccaccgca aacccttccagcttcaacaa gtacatcggt 960 gagtacaagc agaagctgat cagaaagtac cgtttcgtcgtcgagtcttc aggtgaggtc 1020 acagtaaacc gtaacaagtt cgtcgagctg tacaacgagcttacccagat cttcacagag 1080 ttcaactacg ctaagatcta caacgtccag aacaggaagatctacctgtc caacgtgtac 1140 actccggtga cggcgaacat cctggacgac aacgtctacgacatccagaa cggattcaac 1200 atccctaagt ccaacctgaa cgtactattc atgggtcaaaacctgtctcg aaacccagca 1260 ctgcgtaagg tcaaccctga gaacatgctg tacctgttcaccaagttctg ccacaaggca 1320 atcgacggta ga 1332 9 444 PRT ArtificialSequence Synthetic botulinum neurotoxin light chain of serotype C1 basedon wild-type Clostridium botulinum sequence 9 Met Pro Ile Thr Ile AsnAsn Phe Asn Tyr Ser Asp Pro Val Asp Asn 1 5 10 15 Lys Asn Ile Leu TyrLeu Asp Thr His Leu Asn Thr Leu Ala Asn Glu 20 25 30 Pro Glu Lys Ala PheArg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp 35 40 45 Arg Phe Ser Arg AsnSer Asn Pro Asn Leu Asn Lys Pro Pro Arg Val 50 55 60 Thr Ser Pro Lys SerGly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp 65 70 75 80 Ser Asp Lys AspThr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser ArgGlu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr 100 105 110 Asp Ile ProPhe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp 115 120 125 Phe AspVal Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly Asn 130 135 140 AsnTrp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile Thr Gly 145 150 155160 Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr Phe Lys Leu Thr 165170 175 Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala Leu Ser Ile Ile180 185 190 Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn Ala Thr AsnAsp 195 200 205 Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe Cys Met AspPro Ile 210 215 220 Leu Ile Leu Met His Glu Leu Asn His Ala Met His AsnLeu Tyr Gly 225 230 235 240 Ile Ala Ile Pro Asn Asp Gln Thr Ile Ser SerVal Thr Ser Asn Ile 245 250 255 Phe Tyr Ser Gln Tyr Asn Val Lys Leu GluTyr Ala Glu Ile Tyr Ala 260 265 270 Phe Gly Gly Pro Thr Ile Asp Leu IlePro Lys Ser Ala Arg Lys Tyr 275 280 285 Phe Glu Glu Lys Ala Leu Asp TyrTyr Arg Ser Ile Ala Lys Arg Leu 290 295 300 Asn Ser Ile Thr Thr Ala AsnPro Ser Ser Phe Asn Lys Tyr Ile Gly 305 310 315 320 Glu Tyr Lys Gln LysLeu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser 325 330 335 Ser Gly Glu ValThr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn 340 345 350 Glu Leu ThrGln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn 355 360 365 Val GlnAsn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val Thr 370 375 380 AlaAsn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly Phe Asn 385 390 395400 Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly Gln Asn Leu Ser 405410 415 Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn Met Leu Tyr Leu420 425 430 Phe Thr Lys Phe Cys His Lys Ala Ile Asp Gly Arg 435 440 101323 DNA Artificial Sequence Synthetic botulinum neurotoxin light chainof serotype D based on wild-type Clostridium botulinum sequence 10atgacctggc cagtcaagga cttcaactac tccgacccag tcaacgacaa cgacatcttg 60tacttgagaa tcccacaaaa caagttgatc accaccccag tcaaggcttt catgatcacc 120cagaacacct gggttatccc agagagattc tcctccgaca ccaacccatc cctgtccaag 180ccaccaagac caacctccaa gtaccagtct tactacgacc catcttactt gtctaccgac 240gagcaaaagg acaccttctt gaagggtatt atcaagctgt tcaagagaat caacgagaga 300gacatcggta agaagttgat caactacttg gtcgttggtt ccccattcat gggtgactcc 360tctaccccag aggacacctt cgacttcacc agacacacca ccaacattgc cgtcgagaag 420ttcgagaacg gttcctggaa ggtcaccaac atcatcaccc catctgtttt gatcttcggt 480ccattgccaa acatcttgga ctacaccgcc tccctgacct tgcaaggtca gcaatccaac 540ccatccttcg agggtttcgg taccctgtct attttgaagg tcgctccaga gttcttgttg 600accttctccg acgtcacctc caaccaatcc tccgccgtct tgggtaagtc catcttctgt 660atggacccag tcatcgcttt gatgcacgag ttgacccact ccctgcacca gttgtacggt 720attaacatcc catctgacaa gagaatcaga ccacaggtct ctgagggttt cttctcccaa 780gacggtccaa acgttcagtt cgaggagttg tacaccttcg gtggtttgga cgtcgagatt 840atccaaattg agagatccca attgagagag aaggctttgg gtcactacaa ggacatcgcc 900aagagactga acaacatcaa caagaccatt ccatcttcct ggatctccaa cattgacaag 960tacaagaaga ttttctccga gaagtacaac ttcgacaagg acaacaccgg taacttcgtc 1020gttaacatcg acaagttcaa ctctttgtac tccgacttga ccaacgttat gtctgaggtt 1080gtctactcct cccaatacaa cgtcaagaac agaacccact acttctccag acactacttg 1140ccagttttcg ctaacatctt ggacgacaac atttacacca tcagagacgg tttcaacttg 1200accaacaagg gtttcaacat cgagaactcc ggtcaaaaca tcgagagaaa cccagccctg 1260caaaagctgt cctccgagtc tgtcgtcgac ttgttcacca aggtctgttt gagattgacc 1320aag 1323 11 441 PRT Artificial Sequence Synthetic botulinum neurotoxinlight chain of serotype D based on wild-type Clostridium botulinumsequence 11 Met Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val AsnAsp 1 5 10 15 Asn Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu IleThr Thr 20 25 30 Pro Val Lys Ala Phe Met Ile Thr Gln Asn Thr Trp Val IlePro Glu 35 40 45 Arg Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro ProArg Pro 50 55 60 Thr Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu SerThr Asp 65 70 75 80 Glu Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys LeuPhe Lys Arg 85 90 95 Ile Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn TyrLeu Val Val 100 105 110 Gly Ser Pro Phe Met Gly Asp Ser Ser Thr Pro GluAsp Thr Phe Asp 115 120 125 Phe Thr Arg His Thr Thr Asn Ile Ala Val GluLys Phe Glu Asn Gly 130 135 140 Ser Trp Lys Val Thr Asn Ile Ile Thr ProSer Val Leu Ile Phe Gly 145 150 155 160 Pro Leu Pro Asn Ile Leu Asp TyrThr Ala Ser Leu Thr Leu Gln Gly 165 170 175 Gln Gln Ser Asn Pro Ser PheGlu Gly Phe Gly Thr Leu Ser Ile Leu 180 185 190 Lys Val Ala Pro Glu PheLeu Leu Thr Phe Ser Asp Val Thr Ser Asn 195 200 205 Gln Ser Ser Ala ValLeu Gly Lys Ser Ile Phe Cys Met Asp Pro Val 210 215 220 Ile Ala Leu MetHis Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly 225 230 235 240 Ile AsnIle Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly 245 250 255 PhePhe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr 260 265 270Phe Gly Gly Leu Asp Val Glu Ile Ile Gln Ile Glu Arg Ser Gln Leu 275 280285 Arg Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu Asn 290295 300 Asn Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp Lys305 310 315 320 Tyr Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys AspAsn Thr 325 330 335 Gly Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser LeuTyr Ser Asp 340 345 350 Leu Thr Asn Val Met Ser Glu Val Val Tyr Ser SerGln Tyr Asn Val 355 360 365 Lys Asn Arg Thr His Tyr Phe Ser Arg His TyrLeu Pro Val Phe Ala 370 375 380 Asn Ile Leu Asp Asp Asn Ile Tyr Thr IleArg Asp Gly Phe Asn Leu 385 390 395 400 Thr Asn Lys Gly Phe Asn Ile GluAsn Ser Gly Gln Asn Ile Glu Arg 405 410 415 Asn Pro Ala Leu Gln Lys LeuSer Ser Glu Ser Val Val Asp Leu Phe 420 425 430 Thr Lys Val Cys Leu ArgLeu Thr Lys 435 440 12 1266 DNA Artificial Sequence Synthetic botulinumneurotoxin light chain of serotype E based on wild-type Clostridiumbotulinum sequence 12 atgccaaaga ttaactcctt caactacaac gaccctgtcaacgacagaac catcttgtac 60 atcaagccag gcggttgcca ggagttctac aagtccttcaacatcatgaa gaacatctgg 120 atcatccccg agagaaacgt cattggtacc accccccaagacttccaccc ccctacttcc 180 ttgaagaacg gagactccag ttactacgac cctaactacttgcaaagtga cgaggagaag 240 gacagattct tgaagatcgt cacaaagatc ttcaacagaatcaacaacaa cctttcagga 300 ggcatcttgt tggaggagct gtccaaggct aacccatacttgggcaacga caacactcca 360 gataaccagt tccacattgg tgacgcatcc gcagttgagattaagttctc caacggtagc 420 caggacatcc tattgcctaa cgttatcatc atgggagcagagcctgactt gtttgagacc 480 aactcctcca acatctctct acgtaacaac tacatgccaagcaatcacgg tttcggatcc 540 atcgctatcg tcaccttctc ccctgaatat tccttcaggttcaacgacaa cagcatgaac 600 gagttcattc aggatcctgc tctcacgctg atgcacgaattgatccactc cttacatgga 660 ctatatggcg ctaagggcat tactaccaag tacactatcacacagaagca gaacccccta 720 ataaccaaca tccggggtac caacatcgag gagttcttgactttcggagg tactgacttg 780 aacatcatta ctagtgctca gtccaacgac atctacactaaccttctggc tgactacaag 840 aagatcgcgt ctaagcttag caaggtccaa gtctctaacccactgcttaa cccttacaag 900 gacgtcttcg aagcaaagta tggattggac aaggatgctagcggaattta ctcggtcaac 960 atcaacaagt tcaacgacat cttcaagaag ctctacagcttcacggagtt cgacttggcc 1020 accaagttcc aggttaagtg taggcagact tacatcggacagtacaagta cttcaagctg 1080 tccaacctgt tgaacgactc tatctacaac atctcagaaggctacaacat caacaacttg 1140 aaggtcaact tcagaggaca gaatgcaaac ttgaaccctagaatcattac cccaatcacc 1200 ggtagaggac tggtcaagaa gatcatccgt ttctgcaagaacattgtctc tgtcaagggc 1260 atcagg 1266 13 422 PRT Artificial SequenceSynthetic botulinum neurotoxin light chain of serotype E based onwild-type Clostridium botulinum sequence 13 Met Pro Lys Ile Asn Ser PheAsn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile Leu Tyr Ile LysPro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met Lys AsnIle Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro Gln AspPhe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr Tyr AspPro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp Arg Phe Leu LysIle Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu Ser Gly GlyIle Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu Gly AsnAsp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 Ala Ser AlaVal Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 Leu ProAsn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155 160Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165 170175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe 180185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro Ala Leu195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu Tyr GlyAla 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys Gln AsnPro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu Glu PheLeu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser Ala GlnSer Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys Lys IleAla Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu Leu AsnPro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp Lys AspAla Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe Asn AspIle Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu Ala ThrLys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln Tyr LysTyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr Asn IleSer Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 Arg GlyGln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395 400Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405 410415 Ser Val Lys Gly Ile Arg 420 14 1308 DNA Artificial SequenceSynthetic botulinum neurotoxin light chain of serotype F based onwild-type Clostridium botulinum sequence 14 atgccagtcg ctatcaactccttcaactac aacgacccag tcaacgacga caccattttg 60 tacatgcaga tcccatacgaggagaagtct aagaagtact acaaggcttt cgagatcatg 120 agaaacgtct ggattatcgagagaaacacc atcggtacca acccatccga cttcgaccca 180 ccagcctctt tgaagaacggttcctccgct tactacgacc caaactactt gaccaccgac 240 gccgagaagg acagatacttgaagaccacc atcaagttgt tcaagagaat taactctaac 300 ccagccggta aggtcttgttgcaagagatc tcctacgcta agccatacct gggtaacgac 360 cacaccccaa ttgacgagttctccccagtc accagaacca cctccgtcaa catcaagtct 420 accaacgttg agtcctccatgttgttgaac ttgttggttc tgggtgctgg tccagacatt 480 ttcgagtctt gttgttacccagtcagaaag ctgatcgacc cagacgttgt ttacgaccca 540 tctaactacg gtttcggttccattaacatc gttaccttct ctccagagta cgagtacacc 600 ttcaacgaca tctccggtggtcacaactcc tccaccgagt ctttcattgc tgacccagcc 660 atctccctgg ctcacgagctgattcacgct ttgcacggtt tgtacggtgc tagaggtgtc 720 acctacgagg agaccattgaggtcaagcaa gccccattga tgatcgccga gaagccaatc 780 agattggagg agttcttgaccttcggtggt caggacttga acatcatcac ctccgctatg 840 aaggagaaga tctacaacaacctgctggcc aactacgaga agattgccac cagattgtcc 900 gaggtcaact ctgccccaccagagtacgac atcaacgagt acaaggacta cttccaatgg 960 aagtacggtt tggacaagaacgccgacggt tcctacaccg tcaacgagaa caagtccaac 1020 gagatttaca agaagttgtactctttcacc gagtccgacc tggctaacaa gttcaaggtt 1080 aagtgtagaa acacctacttcatcaagtac gagttcttga aggttccaaa cctgttggac 1140 gacgacatct acaccgtttctgagggtttc aacatcggta acttggctgt caacaacaga 1200 ggtcagtcca ttaagctgaacccaaagatc attgactccc cagacaaggg tctggttgag 1260 aagattgtca agttctgtaagtccgtcatc ccaagaaagg gtaccaag 1308 15 436 PRT Artificial SequenceSynthetic botulinum neurotoxin light chain of serotype F based onwild-type Clostridium botulinum sequence 15 Met Pro Val Ala Ile Asn SerPhe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu Tyr MetGln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala Phe GluIle Met Arg Asn Val Trp Ile Ile Glu Arg 35 40 45 Asn Thr Ile Gly Thr AsnPro Ser Asp Phe Asp Pro Pro Ala Ser Leu 50 55 60 Lys Asn Gly Ser Ser AlaTyr Tyr Asp Pro Asn Tyr Leu Thr Thr Asp 65 70 75 80 Ala Glu Lys Asp ArgTyr Leu Lys Thr Thr Ile Lys Leu Phe Lys Arg 85 90 95 Ile Asn Ser Asn ProAla Gly Lys Val Leu Leu Gln Glu Ile Ser Tyr 100 105 110 Ala Lys Pro TyrLeu Gly Asn Asp His Thr Pro Ile Asp Glu Phe Ser 115 120 125 Pro Val ThrArg Thr Thr Ser Val Asn Ile Lys Ser Thr Asn Val Glu 130 135 140 Ser SerMet Leu Leu Asn Leu Leu Val Leu Gly Ala Gly Pro Asp Ile 145 150 155 160Phe Glu Ser Cys Cys Tyr Pro Val Arg Lys Leu Ile Asp Pro Asp Val 165 170175 Val Tyr Asp Pro Ser Asn Tyr Gly Phe Gly Ser Ile Asn Ile Val Thr 180185 190 Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly Gly His195 200 205 Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser LeuAla 210 215 220 His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala ArgGly Val 225 230 235 240 Thr Tyr Glu Glu Thr Ile Glu Val Lys Gln Ala ProLeu Met Ile Ala 245 250 255 Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu ThrPhe Gly Gly Gln Asp 260 265 270 Leu Asn Ile Ile Thr Ser Ala Met Lys GluLys Ile Tyr Asn Asn Leu 275 280 285 Leu Ala Asn Tyr Glu Lys Ile Ala ThrArg Leu Ser Glu Val Asn Ser 290 295 300 Ala Pro Pro Glu Tyr Asp Ile AsnGlu Tyr Lys Asp Tyr Phe Gln Trp 305 310 315 320 Lys Tyr Gly Leu Asp LysAsn Ala Asp Gly Ser Tyr Thr Val Asn Glu 325 330 335 Asn Lys Ser Asn GluIle Tyr Lys Lys Leu Tyr Ser Phe Thr Glu Ser 340 345 350 Asp Leu Ala AsnLys Phe Lys Val Lys Cys Arg Asn Thr Tyr Phe Ile 355 360 365 Lys Tyr GluPhe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp Ile Tyr 370 375 380 Thr ValSer Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn Asn Arg 385 390 395 400Gly Gln Ser Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Pro Asp Lys 405 410415 Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val Ile Pro Arg 420425 430 Lys Gly Thr Lys 435 16 1317 DNA Artificial Sequence Syntheticbotulinum neurotoxin light chain of serotype G based on wild-typeClostridium botulinum sequence 16 atgccagtca acatcaagaa cttcaactacaacgacccaa ttaacaacga cgacatcatg 60 atggagccat tcaacgaccc aggtccaggtacctactaca aggctttcag aatcattgac 120 agaatttgga tcgttccaga gagattcacctacggtttcc aaccagacca gttcaacgcc 180 tccaccggtg tcttctctaa ggacgtctacgagtactacg acccaaccta cttgaagacc 240 gacgctgaga aggacaagtt cttgaagaccatgatcaagt tgttcaacag aattaactct 300 aagccatccg gtcaaagatt gttggacatgattgttgacg ctattccata cttgggtaac 360 gcctccaccc caccagacaa gttcgctgccaacgtcgcta acgtttctat caacaagaag 420 attatccaac caggtgctga ggaccagatcaagggtttga tgaccaactt gattattttc 480 ggtccaggtc cagtcttgtc cgacaacttcaccgactcta tgatcatgaa cggtcactcc 540 ccaatttccg agggtttcgg tgctagaatgatgatcagat tctgtccatc ctgtttgaac 600 gttttcaaca acgtccaaga gaacaaggacacctctatct tctctagaag agcttacttc 660 gctgacccag ctctgaccct gatgcacgagttgatccacg tcttgcacgg tctgtacggt 720 attaagatct ccaacctgcc aattaccccaaacaccaagg agttcttcat gcaacactcc 780 gacccagttc aagccgagga gctgtacaccttcggtggtc acgacccatc tgtttcccca 840 tctaccgaca tgaacattta caacaaggctctgcagaact tccaagacat tgctaacaga 900 ctgaacatcg tctcctctgc ccaaggttctggtatcgaca tttccttgta caagcaaatc 960 tacaagaaca agtacgactt cgtcgaggacccaaacggta agtactctgt tgacaaggac 1020 aagttcgaca agctgtacaa ggctttgatgttcggtttca ccgagaccaa cttggccggt 1080 gagtacggta ttaagaccag atactcttacttctctgagt acctgccacc aatcaagacc 1140 gagaagttgt tggacaacac catctacacccagaacgagg gtttcaacat tgcttccaag 1200 aacttgaaga acgagttcaa cggtcagaacaaggccgtca acaaggaggc ctacgaggag 1260 atttccctgg agcacttggt catctacagaatcgctatgt gtaagccagt catgtac 1317 17 439 PRT Artificial SequenceSynthetic botulinum neurotoxin light chain of serotype G based onwild-type Clostridium botulinum sequence 17 Met Pro Val Asn Ile Lys AsnPhe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15 Asp Asp Ile Met Met GluPro Phe Asn Asp Pro Gly Pro Gly Thr Tyr 20 25 30 Tyr Lys Ala Phe Arg IleIle Asp Arg Ile Trp Ile Val Pro Glu Arg 35 40 45 Phe Thr Tyr Gly Phe GlnPro Asp Gln Phe Asn Ala Ser Thr Gly Val 50 55 60 Phe Ser Lys Asp Val TyrGlu Tyr Tyr Asp Pro Thr Tyr Leu Lys Thr 65 70 75 80 Asp Ala Glu Lys AspLys Phe Leu Lys Thr Met Ile Lys Leu Phe Asn 85 90 95 Arg Ile Asn Ser LysPro Ser Gly Gln Arg Leu Leu Asp Met Ile Val 100 105 110 Asp Ala Ile ProTyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys Phe 115 120 125 Ala Ala AsnVal Ala Asn Val Ser Ile Asn Lys Lys Ile Ile Gln Pro 130 135 140 Gly AlaGlu Asp Gln Ile Lys Gly Leu Met Thr Asn Leu Ile Ile Phe 145 150 155 160Gly Pro Gly Pro Val Leu Ser Asp Asn Phe Thr Asp Ser Met Ile Met 165 170175 Asn Gly His Ser Pro Ile Ser Glu Gly Phe Gly Ala Arg Met Met Ile 180185 190 Arg Phe Cys Pro Ser Cys Leu Asn Val Phe Asn Asn Val Gln Glu Asn195 200 205 Lys Asp Thr Ser Ile Phe Ser Arg Arg Ala Tyr Phe Ala Asp ProAla 210 215 220 Leu Thr Leu Met His Glu Leu Ile His Val Leu His Gly LeuTyr Gly 225 230 235 240 Ile Lys Ile Ser Asn Leu Pro Ile Thr Pro Asn ThrLys Glu Phe Phe 245 250 255 Met Gln His Ser Asp Pro Val Gln Ala Glu GluLeu Tyr Thr Phe Gly 260 265 270 Gly His Asp Pro Ser Val Ser Pro Ser ThrAsp Met Asn Ile Tyr Asn 275 280 285 Lys Ala Leu Gln Asn Phe Gln Asp IleAla Asn Arg Leu Asn Ile Val 290 295 300 Ser Ser Ala Gln Gly Ser Gly IleAsp Ile Ser Leu Tyr Lys Gln Ile 305 310 315 320 Tyr Lys Asn Lys Tyr AspPhe Val Glu Asp Pro Asn Gly Lys Tyr Ser 325 330 335 Val Asp Lys Asp LysPhe Asp Lys Leu Tyr Lys Ala Leu Met Phe Gly 340 345 350 Phe Thr Glu ThrAsn Leu Ala Gly Glu Tyr Gly Ile Lys Thr Arg Tyr 355 360 365 Ser Tyr PheSer Glu Tyr Leu Pro Pro Ile Lys Thr Glu Lys Leu Leu 370 375 380 Asp AsnThr Ile Tyr Thr Gln Asn Glu Gly Phe Asn Ile Ala Ser Lys 385 390 395 400Asn Leu Lys Asn Glu Phe Asn Gly Gln Asn Lys Ala Val Asn Lys Glu 405 410415 Ala Tyr Glu Glu Ile Ser Leu Glu His Leu Val Ile Tyr Arg Ile Ala 420425 430 Met Cys Lys Pro Val Met Tyr 435 18 1239 DNA Artificial SequenceSynthetic N-terminal region of the heavy chain of botulinum neurotoxinserotype A based on wild-type Clostridium botulinum sequence 18atggctctga acgacctgtg catcaaagtt aacaactggg acctgttctt ctccccgtct 60gaagacaact tcactaacga cctgaacaaa ggcgaagaaa tcacctccga cactaacatc 120gaagctgctg aagaaaacat ctctctggac ctgatccagc agtactacct gactttcaac 180ttcgacaacg aaccggaaaa catctccatc gaaaacctgt cttccgacat catcggtcag 240ctggaactga tgccgaacat cgaacgcttc ccgaacggca agaaatacga actggacaaa 300tacaccatgt tccactacct gcgtgctcag gaattcgaac acggtaaatc tcgtatcgct 360ctgactaact ccgttaacga agctctgctg aacccgtctc gcgtttacac cttcttctct 420tccgactacg ttaagaaagt taacaaagct actgaagctg ctatgttcct gggttgggtt 480gaacagctgg tttacgactt caccgacgaa acttctgaag tttccaccac tgacaaaatc 540gctgacatca ctatcatcat cccgtacatc ggcccggctc tgaacatcgg taacatgctg 600tacaaagacg acttcgttgg tgctctgatc ttctctggcg ctgttatcct gctggaattc 660atcccggaaa tcgctatccc ggttctgggt accttcgctc tggtttccta catcgctaac 720aaagttctga ctgttcagac catcgacaac gctctgtcta aacgtaacga aaaatgggac 780gaagtttaca aatacatcgt tactaactgg ctggctaaag ttaacactca gatcgacctg 840atccgtaaga agatgaaaga agctctggaa aaccaggctg aagctactaa agctatcatc 900aactaccagt acaaccagta caccgaagaa gaaaagaaca acatcaactt caacatcgat 960gacctgtcct ctaaactgaa cgaatccatc aacaaagcta tgatcaacat caacaaattc 1020ctgaaccagt gctctgtttc ctacctgatg aactctatga tcccgtacgg cgttaaacgc 1080ctggaagact tcgacgcttc cctgaaagac gctctgctga aatacatccg tgacaactac 1140ggtactctga tcggccaggt tgaccgtctg aaagacaagg ttaacaacac cctgtctact 1200gacatcccgt tccagctgtc caaatacgtt gacaaccag 1239 19 413 PRT ArtificialSequence Synthetic N-terminal region of the heavy chain of botulinumneurotoxin serotype A based on wild-type Clostridium botulinum sequence19 Met Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe 1 510 15 Phe Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu 2025 30 Glu Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser 3540 45 Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu 5055 60 Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln 6570 75 80 Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr85 90 95 Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe100 105 110 Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn GluAla 115 120 125 Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser AspTyr Val 130 135 140 Lys Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe LeuGly Trp Val 145 150 155 160 Glu Gln Leu Val Tyr Asp Phe Thr Asp Glu ThrSer Glu Val Ser Thr 165 170 175 Thr Asp Lys Ile Ala Asp Ile Thr Ile IleIle Pro Tyr Ile Gly Pro 180 185 190 Ala Leu Asn Ile Gly Asn Met Leu TyrLys Asp Asp Phe Val Gly Ala 195 200 205 Leu Ile Phe Ser Gly Ala Val IleLeu Leu Glu Phe Ile Pro Glu Ile 210 215 220 Ala Ile Pro Val Leu Gly ThrPhe Ala Leu Val Ser Tyr Ile Ala Asn 225 230 235 240 Lys Val Leu Thr ValGln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn 245 250 255 Glu Lys Trp AspGlu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala 260 265 270 Lys Val AsnThr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala 275 280 285 Leu GluAsn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr 290 295 300 AsnGln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp 305 310 315320 Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn 325330 335 Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser340 345 350 Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala SerLeu 355 360 365 Lys Asp Ala Leu Leu Lys Tyr Ile Arg Asp Asn Tyr Gly ThrLeu Ile 370 375 380 Gly Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn ThrLeu Ser Thr 385 390 395 400 Asp Ile Pro Phe Gln Leu Ser Lys Tyr Val AspAsn Gln 405 410 20 2583 DNA Artificial Sequence Synthetic polynucleotidesequence for the light chain with Hn of C. botulinum Type A. 20atggttcagt tcgttaacaa acagttcaac tacaaagacc cggttaacgg tgttgacatc 60gcttacatca aaatcccgaa cgttggtcag atgcagccgg ttaaagcatt caaaatccac 120aacaaaatct gggttatccc ggaacgtgac actttcacta acccggaaga aggtgacctg 180aacccgccgc cggaagctaa acaggttccg gtttcttact acgactctac ttacctgtct 240actgacaacg aaaaggacaa ctacctgaaa ggtgttacta aactgtttga acgtatctac 300tctactgacc tgggtcgcat gctgctcact tctatcgttc gtggtatccc gttctggggt 360ggttctacta tcgacactga actgaaagtt atcgacacta actgcatcaa cgttatccag 420ccggacggtt cttaccgttc tgaagaactg aacctggtta tcatcggtcc gtctgctgac 480atcatccagt ttgaatgcaa atctttcggt cacgaagttc tgaacctgac tcgtaacggt 540tacggttcta ctcagtacat ccgtttctct ccggacttca ctttcggttt cgaagaatct 600ctggaagttg acactaaccc gctgctgggt gctggtaaat tcgctactga cccggctgtt 660actctggctc acgaactgat ccacgctggt caccgtctgt acggtatcgc tatcaacccg 720aaccgtgttt tcaaagttaa cactaacgct tactacgaaa tgtctggtct ggaagtttct 780tttgaagaac tgcgtacttt cggtggtcac gacgctaaat tcatcgactc tctgcaggaa 840aacgagttcc gtctgtacta ctactacaaa ttcaaagaca tcgcttctac tctgaacaaa 900gctaaatcta tcgttggtac cactgcttct ctgcagtaca tgaagaacgt tttcaaagaa 960aagtacctgc tgtctgaaga cacttctggt aaattctctg ttgacaaact gaaattcgac 1020aaactgtaca aaatgctgac tgaaatctac actgaagaca acttcgttaa attcttcaaa 1080gttctgaacc gtaaaactta cctgaacttc gacaaagctg ttttcaaaat caacatcgtt 1140ccgaaagtta actacactat ctacgacggt ttcaacctgc gtaacactaa cctggctgct 1200aacttcaacg gtcagaacac tgaaatcaac aacatgaact tcactaaact gaagaacttc 1260actggtctgt ttgagttcta caaactgctg tgcgttcgtg gtatcatcac ttctaaaact 1320aaatctctgg acaaaggtta caacaaagct ctgaacgacc tgtgcatcaa agttaacaac 1380tgggacctgt tcttctcccc gtctgaagac aacttcacta acgacctgaa caaaggcgaa 1440gaaatcacct ccgacactaa catcgaagct gctgaagaaa acatctctct ggacctgatc 1500cagcagtact acctgacttt caacttcgac aacgaaccgg aaaacatctc catcgaaaac 1560ctgtcttccg acatcatcgg tcagctggaa ctgatgccga acatcgaacg cttcccgaac 1620ggcaagaaat acgaactgga caaatacacc atgttccact acctgcgtgc tcaggaattc 1680gaacacggta aatctcgtat cgctctgact aactccgtta acgaagctct gctgaacccg 1740tctcgcgttt acaccttctt ctcttccgac tacgttaaga aagttaacaa agctactgaa 1800gctgctatgt tcctgggttg ggttgaacag ctggtttacg acttcaccga cgaaacttct 1860gaagtttcca ccactgacaa aatcgctgac atcactatca tcatcccgta catcggcccg 1920gctctgaaca tcggtaacat gctgtacaaa gacgacttcg ttggtgctct gatcttctct 1980ggcgctgtta tcctgctgga attcatcccg gaaatcgcta tcccggttct gggtaccttc 2040gctctggttt cctacatcgc taacaaagtt ctgactgttc agaccatcga caacgctctg 2100tctaaacgta acgaaaaatg ggacgaagtt tacaaataca tcgttactaa ctggctggct 2160aaagttaaca ctcagatcga cctgatccgt aagaagatga aagaagctct ggaaaaccag 2220gctgaagcta ctaaagctat catcaactac cagtacaacc agtacaccga agaagaaaag 2280aacaacatca acttcaacat cgatgacctg tcctctaaac tgaacgaatc catcaacaaa 2340gctatgatca acatcaacaa attcctgaac cagtgctctg tttcctacct gatgaactct 2400atgatcccgt acggcgttaa acgcctggaa gacttcgacg cttccctgaa agacgctctg 2460ctgaaataca tccgtgacaa ctacggtact ctgatcggcc aggttgaccg tctgaaagac 2520aaggttaaca acaccctgtc tactgacatc ccgttccagc tgtccaaata cgttgacaac 2580cag 2583 21 861 PRT Artificial Sequence Recombinant protein encoded bySEQ ID NO20 21 Met Val Gln Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp ProVal Asn 1 5 10 15 Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val GlyGln Met Gln 20 25 30 Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp ValIle Pro Glu 35 40 45 Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu AsnPro Pro Pro 50 55 60 Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser ThrTyr Leu Ser 65 70 75 80 Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly ValThr Lys Leu Phe 85 90 95 Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met LeuLeu Thr Ser Ile 100 105 110 Val Arg Gly Ile Pro Phe Trp Gly Gly Ser ThrIle Asp Thr Glu Leu 115 120 125 Lys Val Ile Asp Thr Asn Cys Ile Asn ValIle Gln Pro Asp Gly Ser 130 135 140 Tyr Arg Ser Glu Glu Leu Asn Leu ValIle Ile Gly Pro Ser Ala Asp 145 150 155 160 Ile Ile Gln Phe Glu Cys LysSer Phe Gly His Glu Val Leu Asn Leu 165 170 175 Thr Arg Asn Gly Tyr GlySer Thr Gln Tyr Ile Arg Phe Ser Pro Asp 180 185 190 Phe Thr Phe Gly PheGlu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu 195 200 205 Leu Gly Ala GlyLys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His 210 215 220 Glu Leu IleHis Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro 225 230 235 240 AsnArg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly 245 250 255Leu Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala 260 265270 Lys Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr 275280 285 Tyr Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile290 295 300 Val Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe LysGlu 305 310 315 320 Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe SerVal Asp Lys 325 330 335 Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr GluIle Tyr Thr Glu 340 345 350 Asp Asn Phe Val Lys Phe Phe Lys Val Leu AsnArg Lys Thr Tyr Leu 355 360 365 Asn Phe Asp Lys Ala Val Phe Lys Ile AsnIle Val Pro Lys Val Asn 370 375 380 Tyr Thr Ile Tyr Asp Gly Phe Asn LeuArg Asn Thr Asn Leu Ala Ala 385 390 395 400 Asn Phe Asn Gly Gln Asn ThrGlu Ile Asn Asn Met Asn Phe Thr Lys 405 410 415 Leu Lys Asn Phe Thr GlyLeu Phe Glu Phe Tyr Lys Leu Leu Cys Val 420 425 430 Arg Gly Ile Ile ThrSer Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn 435 440 445 Lys Ala Leu AsnAsp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe 450 455 460 Phe Ser ProSer Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu 465 470 475 480 GluIle Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser 485 490 495Leu Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu 500 505510 Pro Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln 515520 525 Leu Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr530 535 540 Glu Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln GluPhe 545 550 555 560 Glu His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser ValAsn Glu Ala 565 570 575 Leu Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe SerSer Asp Tyr Val 580 585 590 Lys Lys Val Asn Lys Ala Thr Glu Ala Ala MetPhe Leu Gly Trp Val 595 600 605 Glu Gln Leu Val Tyr Asp Phe Thr Asp GluThr Ser Glu Val Ser Thr 610 615 620 Thr Asp Lys Ile Ala Asp Ile Thr IleIle Ile Pro Tyr Ile Gly Pro 625 630 635 640 Ala Leu Asn Ile Gly Asn MetLeu Tyr Lys Asp Asp Phe Val Gly Ala 645 650 655 Leu Ile Phe Ser Gly AlaVal Ile Leu Leu Glu Phe Ile Pro Glu Ile 660 665 670 Ala Ile Pro Val LeuGly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn 675 680 685 Lys Val Leu ThrVal Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn 690 695 700 Glu Lys TrpAsp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala 705 710 715 720 LysVal Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala 725 730 735Leu Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr 740 745750 Asn Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp 755760 765 Asp Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn770 775 780 Ile Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met AsnSer 785 790 795 800 Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe AspAla Ser Leu 805 810 815 Lys Asp Ala Leu Leu Lys Tyr Ile Arg Asp Asn TyrGly Thr Leu Ile 820 825 830 Gly Gln Val Asp Arg Leu Lys Asp Lys Val AsnAsn Thr Leu Ser Thr 835 840 845 Asp Ile Pro Phe Gln Leu Ser Lys Tyr ValAsp Asn Gln 850 855 860 22 1329 DNA Artificial Sequence Syntheticpolynucleotide sequence for the light chain of C. botulinum Type B,optimized for expression in E. coli. 22 atgccagtta ccatcaacaa cttcaactacaacgacccaa tcgacaacaa caacatcatt 60 atgatggagc caccattcgc tagaggtaccggtagatact acaaggcttt caagatcacc 120 gacagaattt ggattattcc agagagatacactttcggtt acaagccaga ggacttcaac 180 aagtcttctg gtattttcaa cagagacgtctgcgagtact acgacccaga ctacctgaac 240 accaacgaca agaagaacat cttcctgcagaccatgatca agctgttcaa cagaatcaag 300 tccaagccat tgggtgagaa gctgctggagatgatcatta acggtatccc atacctgggt 360 gacagaagag tcccactgga ggagttcaacaccaacatcg cctccgtcac cgtcaacaag 420 ctgatctcca acccgggtga ggtcgagcgtaagaagggca tcttcgccaa cctgatcatc 480 ttcggcccag gtccagtctt gaacgagaacgagactattg acattggcat tcaaaaccac 540 ttcgcctcca gagagggttt cggcggtatcatgcaaatga agttctgtcc agagtacgtc 600 tccgttttca acaacgtcca agagaacaagggtgcctcca tcttcaacag aagaggctac 660 ttctccgacc cagccttgat cttgatgcacgagttgatcc acgtcttgca cggtttgtac 720 ggtatcaagg tcgacgactt gccaattgtcccaaacgaga agaagttctt catgcagtcc 780 accgacgcca tccaggccga ggagctgtacaccttcggtg gtcaggaccc atccatcatt 840 accccatcca ccgacaagtc catctacgacaaggtcttgc agaacttcag aggtatcgtc 900 gatagactga acaaggtctt ggtctgcatctccgacccaa acatcaacat caacatttac 960 aagaacaagt tcaaggacaa gtacaagttcgtcgaggact ccgagggtaa gtactccatc 1020 gacgtcgagt ccttcgacaa gctgtacaagtccctgatgt tcggtttcac cgagaccaac 1080 atcgccgaga actacaagat caagaccagagcctcctact tctccgactc cctgccacca 1140 gtcaagatca agaacttgtt ggacaacgaaatctacacta ttgaggaggg tttcaacatt 1200 tccgacaagg acatggagaa ggagtacagaggtcaaaaca aggctattaa caagcaagct 1260 tacgaggaga tttctaagga gcacttggctgtttacaaga ttcaaatgtg taagtctgtt 1320 aagtaatag 1329 23 441 PRTArtificial Sequence Recombinant protein encoded by SEQ ID NO22 23 MetPro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp Pro Ile Asp Asn 1 5 10 15Asn Asn Ile Ile Met Met Glu Pro Pro Phe Ala Arg Gly Thr Gly Arg 20 25 30Tyr Tyr Lys Ala Phe Lys Ile Thr Asp Arg Ile Trp Ile Ile Pro Glu 35 40 45Arg Tyr Thr Phe Gly Tyr Lys Pro Glu Asp Phe Asn Lys Ser Ser Gly 50 55 60Ile Phe Asn Arg Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr Leu Asn 65 70 7580 Thr Asn Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys Leu Phe 85 9095 Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu Met Ile 100105 110 Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu Glu Glu115 120 125 Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu Ile SerAsn 130 135 140 Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe Ala Asn LeuIle Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Asn Glu Asn Glu ThrIle Asp Ile Gly 165 170 175 Ile Gln Asn His Phe Ala Ser Arg Glu Gly PheGly Gly Ile Met Gln 180 185 190 Met Lys Phe Cys Pro Glu Tyr Val Ser ValPhe Asn Asn Val Gln Glu 195 200 205 Asn Lys Gly Ala Ser Ile Phe Asn ArgArg Gly Tyr Phe Ser Asp Pro 210 215 220 Ala Leu Ile Leu Met His Glu LeuIle His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Val Asp AspLeu Pro Ile Val Pro Asn Glu Lys Lys Phe 245 250 255 Phe Met Gln Ser ThrAsp Ala Ile Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly Gln AspPro Ser Ile Ile Thr Pro Ser Thr Asp Lys Ser Ile 275 280 285 Tyr Asp LysVal Leu Gln Asn Phe Arg Gly Ile Val Asp Arg Leu Asn 290 295 300 Lys ValLeu Val Cys Ile Ser Asp Pro Asn Ile Asn Ile Asn Ile Tyr 305 310 315 320Lys Asn Lys Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser Glu Gly 325 330335 Lys Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys Ser Leu 340345 350 Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys Ile Lys355 360 365 Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys IleLys 370 375 380 Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu Gly PheAsn Ile 385 390 395 400 Ser Asp Lys Asp Met Glu Lys Glu Tyr Arg Gly GlnAsn Lys Ala Ile 405 410 415 Asn Lys Gln Ala Tyr Glu Glu Ile Ser Lys GluHis Leu Ala Val Tyr 420 425 430 Lys Ile Gln Met Cys Lys Ser Val Lys 435440 24 2559 DNA Artificial Sequence Synthetic polynucleotide sequencefor the light chain with Hn segment of of C. botulinum Type B, optimizedfor expression in E. coli. 24 atgccagtta ccatcaacaa cttcaactacaacgacccaa tcgacaacaa caacatcatt 60 atgatggagc caccattcgc tagaggtaccggtagatact acaaggcttt caagatcacc 120 gacagaattt ggattattcc agagagatacactttcggtt acaagccaga ggacttcaac 180 aagtcttctg gtattttcaa cagagacgtctgcgagtact acgacccaga ctacctgaac 240 accaacgaca agaagaacat cttcctgcagaccatgatca agctgttcaa cagaatcaag 300 tccaagccat tgggtgagaa gctgctggagatgatcatta acggtatccc atacctgggt 360 gacagaagag tcccactgga ggagttcaacaccaacatcg cctccgtcac cgtcaacaag 420 ctgatctcca acccgggtga ggtcgagcgtaagaagggca tcttcgccaa cctgatcatc 480 ttcggcccag gtccagtctt gaacgagaacgagactattg acattggcat tcaaaaccac 540 ttcgcctcca gagagggttt cggcggtatcatgcaaatga agttctgtcc agagtacgtc 600 tccgttttca acaacgtcca agagaacaagggtgcctcca tcttcaacag aagaggctac 660 ttctccgacc cagccttgat cttgatgcacgagttgatcc acgtcttgca cggtttgtac 720 ggtatcaagg tcgacgactt gccaattgtcccaaacgaga agaagttctt catgcagtcc 780 accgacgcca tccaggccga ggagctgtacaccttcggtg gtcaggaccc atccatcatt 840 accccatcca ccgacaagtc catctacgacaaggtcttgc agaacttcag aggtatcgtc 900 gatagactga acaaggtctt ggtctgcatctccgacccaa acatcaacat caacatttac 960 aagaacaagt tcaaggacaa gtacaagttcgtcgaggact ccgagggtaa gtactccatc 1020 gacgtcgagt ccttcgacaa gctgtacaagtccctgatgt tcggtttcac cgagaccaac 1080 atcgccgaga actacaagat caagaccagagcctcctact tctccgactc cctgccacca 1140 gtcaagatca agaacttgtt ggacaacgaaatctacacta ttgaggaggg tttcaacatt 1200 tccgacaagg acatggagaa ggagtacagaggtcaaaaca aggctattaa caagcaagct 1260 tacgaggaga tttctaagga gcacttggctgtttacaaga ttcaaatgtg taagtctgtt 1320 aaggctccag gaatctgtat cgacgtcgacaacgaggact tgttcttcat cgctgacaag 1380 aactccttct ccgacgactt gtccaagaacgagagaatcg agtacaacac ccagtccaac 1440 tacatcgaga acgacttccc aatcaacgagttgatcttgg acaccgactt gatctccaag 1500 atcgagttgc catccgagaa caccgagtccttgactgact tcaacgtcga cgtcccagtc 1560 tacgagaagc aaccagctat caagaagattttcaccgacg agaacaccat cttccaatac 1620 ctgtactctc agaccttccc tttggacatcagagacatct ccttgacctc ttccttcgac 1680 gacgccctgc tgttctccaa caaggtctactccttcttct ccatggacta catcaagact 1740 gctaacaagg tcgtcgaggc cggtttgttcgctggttggg tcaagcagat cgtcaacgat 1800 ttcgtcatcg aggctaacaa gtccaacaccatggacaaga ttgccgacat ctccttgatt 1860 gtcccataca tcggtttggc cttgaacgtcggtaacgaga ccgccaaggg taacttcgag 1920 aacgctttcg agatcgctgg tgcctccatcttgttggagt tcatcccaga gttgttgatc 1980 ccagtcgtcg gtgccttctt gttggagtcctacatcgaca acaagaacaa gatcatcaag 2040 accatcgaca acgctttgac caagagaaacgagaagtggt ccgacatgta cggtttgatc 2100 gtcgcccaat ggttgtccac cgtcaacacccaattctaca ccatcaagga gggtatgtac 2160 aaggccttga actaccaggc ccaagctttggaggagatca tcaagtacag atacaacatc 2220 tactccgaga aggagaagtc caacattaacatcgacttca acgacatcaa ctccaagctg 2280 aacgagggta ttaaccaggc catcgacaacatcaacaact tcatcaacgg ttgttccgtc 2340 tcctacttga tgaagaagat gattccattggccgtcgaga agttgttgga cttcgacaac 2400 accctgaaga agaacttgtt gaactacatcgacgagaaca agttgtactt gatcggttcc 2460 gctgagtacg agaagtccaa ggtcaacaagtacttgaaga ccatcatgcc attcgacttg 2520 tccatctaca ccaacgacac catcttgatcgagatgttc 2559 25 852 PRT Artificial Sequence Recombinant proteinencoded by SEQ ID NO24 25 Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn AspPro Ile Asp Asn Asn 1 5 10 15 Asn Ile Ile Met Met Glu Pro Pro Phe AlaArg Gly Thr Gly Arg Tyr 20 25 30 Tyr Lys Ala Phe Lys Ile Thr Asp Arg IleTrp Ile Ile Pro Glu Arg 35 40 45 Tyr Thr Phe Gly Tyr Lys Pro Glu Asp PheAsn Lys Ser Ser Gly Ile 50 55 60 Phe Asn Arg Asp Val Cys Glu Tyr Tyr AspPro Asp Tyr Leu Asn Thr 65 70 75 80 Asn Asp Lys Lys Asn Ile Phe Leu GlnThr Met Ile Lys Leu Phe Asn 85 90 95 Arg Ile Lys Ser Lys Pro Leu Gly GluLys Leu Leu Glu Met Ile Ile 100 105 110 Asn Gly Ile Pro Tyr Leu Gly AspArg Arg Val Pro Leu Glu Glu Phe 115 120 125 Asn Thr Asn Ile Ala Ser ValThr Val Asn Lys Leu Ile Ser Asn Pro 130 135 140 Gly Glu Val Glu Arg LysLys Gly Ile Phe Ala Asn Leu Ile Ile Phe 145 150 155 160 Gly Pro Gly ProVal Leu Asn Glu Asn Glu Thr Ile Asp Ile Gly Ile 165 170 175 Gln Asn HisPhe Ala Ser Arg Glu Gly Phe Gly Gly Ile Met Gln Met 180 185 190 Lys PheCys Pro Glu Tyr Val Ser Val Phe Asn Asn Val Gln Glu Asn 195 200 205 LysGly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser Asp Pro Ala 210 215 220Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly Leu Tyr Gly 225 230235 240 Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys Lys Phe Phe245 250 255 Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu Leu Tyr Thr PheGly 260 265 270 Gly Gln Asp Pro Ser Ile Ile Thr Pro Ser Thr Asp Lys SerIle Tyr 275 280 285 Asp Lys Val Leu Gln Asn Phe Arg Gly Ile Val Asp ArgLeu Asn Lys 290 295 300 Val Leu Val Cys Ile Ser Asp Pro Asn Ile Asn IleAsn Ile Tyr Lys 305 310 315 320 Asn Lys Phe Lys Asp Lys Tyr Lys Phe ValGlu Asp Ser Glu Gly Lys 325 330 335 Tyr Ser Ile Asp Val Glu Ser Phe AspLys Leu Tyr Lys Ser Leu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn IleAla Glu Asn Tyr Lys Ile Lys Thr 355 360 365 Arg Ala Ser Tyr Phe Ser AspSer Leu Pro Pro Val Lys Ile Lys Asn 370 375 380 Leu Leu Asp Asn Glu IleTyr Thr Ile Glu Glu Gly Phe Asn Ile Ser 385 390 395 400 Asp Lys Asp MetGlu Lys Glu Tyr Arg Gly Gln Asn Lys Ala Ile Asn 405 410 415 Lys Gln AlaTyr Glu Glu Ile Ser Lys Glu His Leu Ala Val Tyr Lys 420 425 430 Ile GlnMet Cys Lys Ser Val Lys Ala Pro Gly Ile Cys Ile Asp Val 435 440 445 AspAsn Glu Asp Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser Asp 450 455 460Asp Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn Tyr 465 470475 480 Ile Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp Leu485 490 495 Ile Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu ThrAsp 500 505 510 Phe Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro Ala IleLys Lys 515 520 525 Ile Phe Thr Asp Glu Asn Thr Ile Phe Gln Tyr Leu TyrSer Gln Thr 530 535 540 Phe Pro Leu Asp Ile Arg Asp Ile Ser Leu Thr SerSer Phe Asp Asp 545 550 555 560 Ala Leu Leu Phe Ser Asn Lys Val Tyr SerPhe Phe Ser Met Asp Tyr 565 570 575 Ile Lys Thr Ala Asn Lys Val Val GluAla Gly Leu Phe Ala Gly Trp 580 585 590 Val Lys Gln Ile Val Asn Asp PheVal Ile Glu Ala Asn Lys Ser Asn 595 600 605 Thr Met Asp Lys Ile Ala AspIle Ser Leu Ile Val Pro Tyr Ile Gly 610 615 620 Leu Ala Leu Asn Val GlyAsn Glu Thr Ala Lys Gly Asn Phe Glu Asn 625 630 635 640 Ala Phe Glu IleAla Gly Ala Ser Ile Leu Leu Glu Phe Ile Pro Glu 645 650 655 Leu Leu IlePro Val Val Gly Ala Phe Leu Leu Glu Ser Tyr Ile Asp 660 665 670 Asn LysAsn Lys Ile Ile Lys Thr Ile Asp Asn Ala Leu Thr Lys Arg 675 680 685 AsnGlu Lys Trp Ser Asp Met Tyr Gly Leu Ile Val Ala Gln Trp Leu 690 695 700Ser Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr Lys 705 710715 720 Ala Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr Arg725 730 735 Tyr Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile AspPhe 740 745 750 Asn Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln AlaIle Asp 755 760 765 Asn Ile Asn Asn Phe Ile Asn Gly Cys Ser Val Ser TyrLeu Met Lys 770 775 780 Lys Met Ile Pro Leu Ala Val Glu Lys Leu Leu AspPhe Asp Asn Thr 785 790 795 800 Leu Lys Lys Asn Leu Leu Asn Tyr Ile AspGlu Asn Lys Leu Tyr Leu 805 810 815 Ile Gly Ser Ala Glu Tyr Glu Lys SerLys Val Asn Lys Tyr Leu Lys 820 825 830 Thr Ile Met Pro Phe Asp Leu SerIle Tyr Thr Asn Asp Thr Ile Leu 835 840 845 Ile Glu Met Phe 850 26 1311DNA Artificial Sequence Synthetic polynucleotide sequence for the lightchain of of C. botulinum Type C, optimized for expression in E. coli. 26atgccaatca ccatcaacaa cttcaactac tcagaccctg tcgacaacaa gaacattctg 60tacctggaca ctcacctgaa caccctagct aacgagcctg agaaggcctt tcggatcacc 120ggaaacatct gggtcatccc tgatcgtttc tcccgtaact ccaaccccaa cctgaacaag 180cctcctcggg tcaccagccc taagagtggt tactacgacc ctaactacct gagtaccgac 240tctgacaagg acaccttcct gaaggagatc atcaagctgt tcaagcgtat caactcccgt 300gagatcggag aggagctcat ctacagactt tcgaccgata tccccttccc tggtaacaac 360aatactccaa tcaacacctt cgacttcgac gtcgacttca actccgtcga cgtcaagact 420cggcagggta acaactgggt taagactggt agcatcaacc cttccgtcat catcactgga 480cctcgtgaga acatcatcga cccagagact tccacgttca agctgactaa caacaccttc 540gcggctcaag aaggattcgg tgctctgtca atcatctcca tctcacctcg tttcatgctg 600acctactcga acgcaaccaa cgacgtcgga gagggtaggt tctctaagtc tgagttctgc 660atggacccaa tcctgatcct gatgcatgag ctgaaccatg caatgcacaa cctgtacgga 720atcgctatcc caaacgacca gaccatctcc tccgtgacct ccaacatctt ctactcccag 780tacaacgtga agctggagta cgcagagatc tacgctttcg gaggtccaac tatcgacctt 840atccctaagt ccgctaggaa gtacttcgag gagaaggctt tggattacta cagatccatc 900gctaagagac tgaacagtat caccaccgca aacccttcca gcttcaacaa gtacatcggt 960gagtacaagc agaagctgat cagaaagtac cgtttcgtcg tcgagtcttc aggtgaggtc 1020acagtaaacc gtaacaagtt cgtcgagctg tacaacgagc ttacccagat cttcacagag 1080ttcaactacg ctaagatcta caacgtccag aacaggaaga tctacctgtc caacgtgtac 1140actccggtga cggcgaacat cctggacgac aacgtctacg acatccagaa cggattcaac 1200atccctaagt ccaacctgaa cgtactattc atgggtcaaa acctgtctcg aaacccagca 1260ctgcgtaagg tcaaccctga gaacatgctg tacctgttca ccaagttctg c 1311 27 436 PRTArtificial Sequence Recombinant protein encoded by SEQ ID NO26 27 ProIle Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn Lys 1 5 10 15Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn Glu Pro 20 25 30Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro Asp Arg 35 40 45Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg Val Thr 50 55 60Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr Asp Ser 65 70 7580 Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe Lys Arg Ile 85 9095 Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu Ser Thr Asp 100105 110 Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn Thr Phe Asp Phe115 120 125 Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr Arg Gln Gly AsnAsn 130 135 140 Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val Ile Ile ThrGly Pro 145 150 155 160 Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr PheLys Leu Thr Asn 165 170 175 Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly AlaLeu Ser Ile Ile Ser 180 185 190 Ile Ser Pro Arg Phe Met Leu Thr Tyr SerAsn Ala Thr Asn Asp Val 195 200 205 Gly Glu Gly Arg Phe Ser Lys Ser GluPhe Cys Met Asp Pro Ile Leu 210 215 220 Ile Leu Met His Glu Leu Asn HisAla Met His Asn Leu Tyr Gly Ile 225 230 235 240 Ala Ile Pro Asn Asp GlnThr Ile Ser Ser Val Thr Ser Asn Ile Phe 245 250 255 Tyr Ser Gln Tyr AsnVal Lys Leu Glu Tyr Ala Glu Ile Tyr Ala Phe 260 265 270 Gly Gly Pro ThrIle Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr Phe 275 280 285 Glu Glu LysAla Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu Asn 290 295 300 Ser IleThr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly Glu 305 310 315 320Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu Ser Ser 325 330335 Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu Tyr Asn Glu 340345 350 Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys Ile Tyr Asn Val355 360 365 Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr Thr Pro Val ThrAla 370 375 380 Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln Asn Gly PheAsn Ile 385 390 395 400 Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly GlnAsn Leu Ser Arg 405 410 415 Asn Pro Ala Leu Arg Lys Val Asn Pro Glu AsnMet Leu Tyr Leu Phe 420 425 430 Thr Lys Phe Cys 435 28 2436 DNAArtificial Sequence Synthetic polynucleotide sequence for the lightchain with Hn segment of of C. botulinum Type C, optimized forexpression in E. coli. 28 atgccaatca ccatcaacaa cttcaactac tcagaccctgtcgacaacaa gaacattctg 60 tacctggaca ctcacctgaa caccctagct aacgagcctgagaaggcctt tcggatcacc 120 ggaaacatct gggtcatccc tgatcgtttc tcccgtaactccaaccccaa cctgaacaag 180 cctcctcggg tcaccagccc taagagtggt tactacgaccctaactacct gagtaccgac 240 tctgacaagg acaccttcct gaaggagatc atcaagctgttcaagcgtat caactcccgt 300 gagatcggag aggagctcat ctacagactt tcgaccgatatccccttccc tggtaacaac 360 aatactccaa tcaacacctt cgacttcgac gtcgacttcaactccgtcga cgtcaagact 420 cggcagggta acaactgggt taagactggt agcatcaacccttccgtcat catcactgga 480 cctcgtgaga acatcatcga cccagagact tccacgttcaagctgactaa caacaccttc 540 gcggctcaag aaggattcgg tgctctgtca atcatctccatctcacctcg tttcatgctg 600 acctactcga acgcaaccaa cgacgtcgga gagggtaggttctctaagtc tgagttctgc 660 atggacccaa tcctgatcct gatgcatgag ctgaaccatgcaatgcacaa cctgtacgga 720 atcgctatcc caaacgacca gaccatctcc tccgtgacctccaacatctt ctactcccag 780 tacaacgtga agctggagta cgcagagatc tacgctttcggaggtccaac tatcgacctt 840 atccctaagt ccgctaggaa gtacttcgag gagaaggctttggattacta cagatccatc 900 gctaagagac tgaacagtat caccaccgca aacccttccagcttcaacaa gtacatcggt 960 gagtacaagc agaagctgat cagaaagtac cgtttcgtcgtcgagtcttc aggtgaggtc 1020 acagtaaacc gtaacaagtt cgtcgagctg tacaacgagcttacccagat cttcacagag 1080 ttcaactacg ctaagatcta caacgtccag aacaggaagatctacctgtc caacgtgtac 1140 actccggtga cggcgaacat cctggacgac aacgtctacgacatccagaa cggattcaac 1200 atccctaagt ccaacctgaa cgtactattc atgggtcaaaacctgtctcg aaacccagca 1260 ctgcgtaagg tcaaccctga gaacatgctg tacctgttcaccaagttctg ctccctgtac 1320 aacaagaccc ttgactgtag agagctgctg gtgaagaacactgacctgcc attcatcggt 1380 gacatcagtg acgtgaagac tgacatcttc ctgcgtaaggacatcaacga ggagactgag 1440 gtgatctact acccagacaa cgtgtcagta gaccaagtgatcctcagtaa gaacacctcc 1500 gagcatggac aactagacct gctctaccct agtatcgacagtgagagtga gatcctgcca 1560 ggggagaatc aagtcttcta cgacaaccgt acccagaacgtggactacct gaactcctac 1620 tactacctag agtctcagaa gctgagtgac aacgtggaggacttcacttt cacgcgttca 1680 atcgaggagg ctctggacaa cagtgcaaag gtgtacacttacttccctac cctggctaac 1740 aaggtgaatg ccggtgtgca aggtggtctg ttcctgatgtgggcaaacga cgtggttgag 1800 gacttcacta ccaacatcct gcgtaaggac acactggacaagatctcaga tgtgtcagct 1860 atcatcccct acatcggacc cgcactgaac atctccaactctgtgcgtcg tggaaacttc 1920 actgaggcat tcgcagtcac tggtgtcacc atcctgctggaggcattccc tgagttcaca 1980 atccctgctc tgggtgcatt cgtgatctac agtaaggtccaggagcgaaa cgagatcatc 2040 aagaccatcg acaactgtct ggagcagagg atcaagagatggaaggactc ctacgagtgg 2100 atgatgggaa cgtggttgtc caggatcatc acccagttcaacaacatctc ctaccagatg 2160 tacgactccc tgaactacca ggcaggtgca atcaaggctaagatcgacct ggagtacaag 2220 aagtactccg gaagcgacaa ggagaacatc aagagccaggttgagaacct gaagaacagt 2280 ctggacgtca agatctcgga ggcaatgaac aacatcaacaagttcatccg agagtgctcc 2340 gtcacctacc tgttcaagaa catgctgcct aaggtcatcgacgagctgaa cgagttcgac 2400 cgaaacacca aggcaaagct gatcaacctg atcgac 243629 811 PRT Artificial Sequence Recombinant protein encoded by SEQ IDNO28 29 Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp Pro Val Asp Asn Lys1 5 10 15 Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr Leu Ala Asn GluPro 20 25 30 Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp Val Ile Pro AspArg 35 40 45 Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys Pro Pro Arg ValThr 50 55 60 Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr Leu Ser Thr AspSer 65 70 75 80 Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile Lys Leu Phe LysArg Ile 85 90 95 Asn Ser Arg Glu Ile Gly Glu Glu Leu Ile Tyr Arg Leu SerThr Asp 100 105 110 Ile Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile Asn ThrPhe Asp Phe 115 120 125 Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr ArgGln Gly Asn Asn 130 135 140 Trp Val Lys Thr Gly Ser Ile Asn Pro Ser ValIle Ile Thr Gly Pro 145 150 155 160 Arg Glu Asn Ile Ile Asp Pro Glu ThrSer Thr Phe Lys Leu Thr Asn 165 170 175 Asn Thr Phe Ala Ala Gln Glu GlyPhe Gly Ala Leu Ser Ile Ile Ser 180 185 190 Ile Ser Pro Arg Phe Met LeuThr Tyr Ser Asn Ala Thr Asn Asp Val 195 200 205 Gly Glu Gly Arg Phe SerLys Ser Glu Phe Cys Met Asp Pro Ile Leu 210 215 220 Ile Leu Met His GluLeu Asn His Ala Met His Asn Leu Tyr Gly Ile 225 230 235 240 Ala Ile ProAsn Asp Gln Thr Ile Ser Ser Val Thr Ser Asn Ile Phe 245 250 255 Tyr SerGln Tyr Asn Val Lys Leu Glu Tyr Ala Glu Ile Tyr Ala Phe 260 265 270 GlyGly Pro Thr Ile Asp Leu Ile Pro Lys Ser Ala Arg Lys Tyr Phe 275 280 285Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile Ala Lys Arg Leu Asn 290 295300 Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn Lys Tyr Ile Gly Glu 305310 315 320 Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg Phe Val Val Glu SerSer 325 330 335 Gly Glu Val Thr Val Asn Arg Asn Lys Phe Val Glu Leu TyrAsn Glu 340 345 350 Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala Lys IleTyr Asn Val 355 360 365 Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr ThrPro Val Thr Ala 370 375 380 Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile GlnAsn Gly Phe Asn Ile 385 390 395 400 Pro Lys Ser Asn Leu Asn Val Leu PheMet Gly Gln Asn Leu Ser Arg 405 410 415 Asn Pro Ala Leu Arg Lys Val AsnPro Glu Asn Met Leu Tyr Leu Phe 420 425 430 Thr Lys Phe Cys Ser Leu TyrAsn Lys Thr Leu Asp Cys Arg Glu Leu 435 440 445 Leu Val Lys Asn Thr AspLeu Pro Phe Ile Gly Asp Ile Ser Asp Val 450 455 460 Lys Thr Asp Ile PheLeu Arg Lys Asp Ile Asn Glu Glu Thr Glu Val 465 470 475 480 Ile Tyr TyrPro Asp Asn Val Ser Val Asp Gln Val Ile Leu Ser Lys 485 490 495 Asn ThrSer Glu His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp 500 505 510 SerGlu Ser Glu Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn 515 520 525Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser 530 535540 Gln Lys Leu Ser Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile 545550 555 560 Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe ProThr 565 570 575 Leu Ala Asn Lys Val Asn Ala Gly Val Gln Gly Gly Leu PheLeu Met 580 585 590 Trp Ala Asn Asp Val Val Glu Asp Phe Thr Thr Asn IleLeu Arg Lys 595 600 605 Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala IleIle Pro Tyr Ile 610 615 620 Gly Pro Ala Leu Asn Ile Ser Asn Ser Val ArgArg Gly Asn Phe Thr 625 630 635 640 Glu Ala Phe Ala Val Thr Gly Val ThrIle Leu Leu Glu Ala Phe Pro 645 650 655 Glu Phe Thr Ile Pro Ala Leu GlyAla Phe Val Ile Tyr Ser Lys Val 660 665 670 Gln Glu Arg Asn Glu Ile IleLys Thr Ile Asp Asn Cys Leu Glu Gln 675 680 685 Arg Ile Lys Arg Trp LysAsp Ser Tyr Glu Trp Met Met Gly Thr Trp 690 695 700 Leu Ser Arg Ile IleThr Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr 705 710 715 720 Asp Ser LeuAsn Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu 725 730 735 Glu TyrLys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln 740 745 750 ValGlu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met 755 760 765Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe 770 775780 Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg 785790 795 800 Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile Asp 805 810 30 1323DNA Artificial Sequence Synthetic polynucleotide sequence for the lightchain of of C. botulinum Type D, optimized for expression in E. coli. 30atgacctggc cagtcaagga cttcaactac tccgacccag tcaacgacaa cgacatcttg 60tacttgagaa tcccacaaaa caagttgatc accaccccag tcaaggcttt catgatcacc 120cagaacacct gggttatccc agagagattc tcctccgaca ccaacccatc cctgtccaag 180ccaccaagac caacctccaa gtaccagtct tactacgacc catcttactt gtctaccgac 240gagcaaaagg acaccttctt gaagggtatt atcaagctgt tcaagagaat caacgagaga 300gacatcggta agaagttgat caactacttg gtcgttggtt ccccattcat gggtgactcc 360tctaccccag aggacacctt cgacttcacc agacacacca ccaacattgc cgtcgagaag 420ttcgagaacg gttcctggaa ggtcaccaac atcatcaccc catctgtttt gatcttcggt 480ccattgccaa acatcttgga ctacaccgcc tccctgacct tgcaaggtca gcaatccaac 540ccatccttcg agggtttcgg taccctgtct attttgaagg tcgctccaga gttcttgttg 600accttctccg acgtcacctc caaccaatcc tccgccgtct tgggtaagtc catcttctgt 660atggacccag tcatcgcttt gatgcacgag ttgacccact ccctgcacca gttgtacggt 720attaacatcc catctgacaa gagaatcaga ccacaggtct ctgagggttt cttctcccaa 780gacggtccaa acgttcagtt cgaggagttg tacaccttcg gtggtttgga cgtcgagatt 840atccaaattg agagatccca attgagagag aaggctttgg gtcactacaa ggacatcgcc 900aagagactga acaacatcaa caagaccatt ccatcttcct ggatctccaa cattgacaag 960tacaagaaga ttttctccga gaagtacaac ttcgacaagg acaacaccgg taacttcgtc 1020gttaacatcg acaagttcaa ctctttgtac tccgacttga ccaacgttat gtctgaggtt 1080gtctactcct cccaatacaa cgtcaagaac agaacccact acttctccag acactacttg 1140ccagttttcg ctaacatctt ggacgacaac atttacacca tcagagacgg tttcaacttg 1200accaacaagg gtttcaacat cgagaactcc ggtcaaaaca tcgagagaaa cccagccctg 1260caaaagctgt cctccgagtc tgtcgtcgac ttgttcacca aggtctgttt gagattgacc 1320aag 1323 31 440 PRT Artificial Sequence Recombinant protein encoded bySEQ ID NO30 31 Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val AsnAsp Asn 1 5 10 15 Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu IleThr Thr Pro 20 25 30 Val Lys Ala Phe Met Ile Thr Gln Asn Thr Trp Val IlePro Glu Arg 35 40 45 Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro ProArg Pro Thr 50 55 60 Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu SerThr Asp Glu 65 70 75 80 Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys LeuPhe Lys Arg Ile 85 90 95 Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn TyrLeu Val Val Gly 100 105 110 Ser Pro Phe Met Gly Asp Ser Ser Thr Pro GluAsp Thr Phe Asp Phe 115 120 125 Thr Arg His Thr Thr Asn Ile Ala Val GluLys Phe Glu Asn Gly Ser 130 135 140 Trp Lys Val Thr Asn Ile Ile Thr ProSer Val Leu Ile Phe Gly Pro 145 150 155 160 Leu Pro Asn Ile Leu Asp TyrThr Ala Ser Leu Thr Leu Gln Gly Gln 165 170 175 Gln Ser Asn Pro Ser PheGlu Gly Phe Gly Thr Leu Ser Ile Leu Lys 180 185 190 Val Ala Pro Glu PheLeu Leu Thr Phe Ser Asp Val Thr Ser Asn Gln 195 200 205 Ser Ser Ala ValLeu Gly Lys Ser Ile Phe Cys Met Asp Pro Val Ile 210 215 220 Ala Leu MetHis Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly Ile 225 230 235 240 AsnIle Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly Phe 245 250 255Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr Phe 260 265270 Gly Gly Leu Asp Val Glu Ile Ile Gln Ile Glu Arg Ser Gln Leu Arg 275280 285 Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu Asn Asn290 295 300 Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp LysTyr 305 310 315 320 Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys AspAsn Thr Gly 325 330 335 Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser LeuTyr Ser Asp Leu 340 345 350 Thr Asn Val Met Ser Glu Val Val Tyr Ser SerGln Tyr Asn Val Lys 355 360 365 Asn Arg Thr His Tyr Phe Ser Arg His TyrLeu Pro Val Phe Ala Asn 370 375 380 Ile Leu Asp Asp Asn Ile Tyr Thr IleArg Asp Gly Phe Asn Leu Thr 385 390 395 400 Asn Lys Gly Phe Asn Ile GluAsn Ser Gly Gln Asn Ile Glu Arg Asn 405 410 415 Pro Ala Leu Gln Lys LeuSer Ser Glu Ser Val Val Asp Leu Phe Thr 420 425 430 Lys Val Cys Leu ArgLeu Thr Lys 435 440 32 2475 DNA Artificial Sequence Syntheticpolynucleotide sequence for the light chain with Hn segment of of C.botulinum Type D, optimized for expression in E. coli. 32 atgacctggccagtcaagga cttcaactac tccgacccag tcaacgacaa cgacatcttg 60 tacttgagaatcccacaaaa caagttgatc accaccccag tcaaggcttt catgatcacc 120 cagaacacctgggttatccc agagagattc tcctccgaca ccaacccatc cctgtccaag 180 ccaccaagaccaacctccaa gtaccagtct tactacgacc catcttactt gtctaccgac 240 gagcaaaaggacaccttctt gaagggtatt atcaagctgt tcaagagaat caacgagaga 300 gacatcggtaagaagttgat caactacttg gtcgttggtt ccccattcat gggtgactcc 360 tctaccccagaggacacctt cgacttcacc agacacacca ccaacattgc cgtcgagaag 420 ttcgagaacggttcctggaa ggtcaccaac atcatcaccc catctgtttt gatcttcggt 480 ccattgccaaacatcttgga ctacaccgcc tccctgacct tgcaaggtca gcaatccaac 540 ccatccttcgagggtttcgg taccctgtct attttgaagg tcgctccaga gttcttgttg 600 accttctccgacgtcacctc caaccaatcc tccgccgtct tgggtaagtc catcttctgt 660 atggacccagtcatcgcttt gatgcacgag ttgacccact ccctgcacca gttgtacggt 720 attaacatcccatctgacaa gagaatcaga ccacaggtct ctgagggttt cttctcccaa 780 gacggtccaaacgttcagtt cgaggagttg tacaccttcg gtggtttgga cgtcgagatt 840 atccaaattgagagatccca attgagagag aaggctttgg gtcactacaa ggacatcgcc 900 aagagactgaacaacatcaa caagaccatt ccatcttcct ggatctccaa cattgacaag 960 tacaagaagattttctccga gaagtacaac ttcgacaagg acaacaccgg taacttcgtc 1020 gttaacatcgacaagttcaa ctctttgtac tccgacttga ccaacgttat gtctgaggtt 1080 gtctactcctcccaatacaa cgtcaagaac agaacccact acttctccag acactacttg 1140 ccagttttcgctaacatctt ggacgacaac atttacacca tcagagacgg tttcaacttg 1200 accaacaagggtttcaacat cgagaactcc ggtcaaaaca tcgagagaaa cccagccctg 1260 caaaagctgtcctccgagtc tgtcgtcgac ttgttcacca aggtctgttt gagattgacc 1320 aagaactcccgtgacgactc cacctgcatc aaggtcaaga acaacagact gccatacgtt 1380 gccgacaaggactccatctc ccaggagatc ttcgagaaca agatcatcac cgacgagacc 1440 aacgttcaaaactactccga caagttctct ttggacgagt ccatcctgga cggtcaggtc 1500 ccaatcaacccagagatcgt cgacccactg ttgccaaacg tcaacatgga gccattgaac 1560 ttgccaggtgaggagatcgt cttctacgac gacatcacca agtacgtcga ctacttgaac 1620 tcctactactacttggagtc tcaaaagttg tctaacaacg tcgagaacat caccttgacc 1680 acctccgtcgaggaggcctt gggttactct aacaagatct acaccttcct gccatccttg 1740 gctgagaaggttaacaaggg tgttcaagct ggtttgttcc tgaactgggc caacgaggtc 1800 gtcgaggacttcaccaccaa catcatgaag aaggacaccc tggacaagat ctccgacgtc 1860 tccgtcatcatcccatacat cggtccagcc ttgaacatcg gtaactccgc cctgagaggt 1920 aacttcaaccaggccttcgc caccgccggt gtcgccttcc tgctggaggg tttcccagag 1980 ttcaccatcccagccctggg tgtcttcacc ttctactcct ccatccagga gagagagaag 2040 atcatcaagaccatcgagaa ctgcttggag cagagagtca agagatggaa ggactcctac 2100 cagtggatggtttccaactg gctgtccaga atcaccaccc aattcaacca catcaactac 2160 cagatgtacgactccctgtc ctaccaggcc gacgccatca aggccaagat cgacctggag 2220 tacaagaagtactccggttc cgacaaggag aacatcaagt cccaggtcga gaacctgaag 2280 aactccttggacgtcaagat ctccgaggcc atgaacaaca tcaacaagtt catccgtgag 2340 tgttccgtcacctacctgtt caagaacatg ctgccaaagg tcatcgacga gctgaacaag 2400 ttcgacctgagaaccaagac cgagctgatc aacctgatcg actcccacaa catcatcctg 2460 gttggtgaggttgac 2475 33 824 PRT Artificial Sequence Recombinant protein encoded bySEQ ID NO32 33 Thr Trp Pro Val Lys Asp Phe Asn Tyr Ser Asp Pro Val AsnAsp Asn 1 5 10 15 Asp Ile Leu Tyr Leu Arg Ile Pro Gln Asn Lys Leu IleThr Thr Pro 20 25 30 Val Lys Ala Phe Met Ile Thr Gln Asn Thr Trp Val IlePro Glu Arg 35 40 45 Phe Ser Ser Asp Thr Asn Pro Ser Leu Ser Lys Pro ProArg Pro Thr 50 55 60 Ser Lys Tyr Gln Ser Tyr Tyr Asp Pro Ser Tyr Leu SerThr Asp Glu 65 70 75 80 Gln Lys Asp Thr Phe Leu Lys Gly Ile Ile Lys LeuPhe Lys Arg Ile 85 90 95 Asn Glu Arg Asp Ile Gly Lys Lys Leu Ile Asn TyrLeu Val Val Gly 100 105 110 Ser Pro Phe Met Gly Asp Ser Ser Thr Pro GluAsp Thr Phe Asp Phe 115 120 125 Thr Arg His Thr Thr Asn Ile Ala Val GluLys Phe Glu Asn Gly Ser 130 135 140 Trp Lys Val Thr Asn Ile Ile Thr ProSer Val Leu Ile Phe Gly Pro 145 150 155 160 Leu Pro Asn Ile Leu Asp TyrThr Ala Ser Leu Thr Leu Gln Gly Gln 165 170 175 Gln Ser Asn Pro Ser PheGlu Gly Phe Gly Thr Leu Ser Ile Leu Lys 180 185 190 Val Ala Pro Glu PheLeu Leu Thr Phe Ser Asp Val Thr Ser Asn Gln 195 200 205 Ser Ser Ala ValLeu Gly Lys Ser Ile Phe Cys Met Asp Pro Val Ile 210 215 220 Ala Leu MetHis Glu Leu Thr His Ser Leu His Gln Leu Tyr Gly Ile 225 230 235 240 AsnIle Pro Ser Asp Lys Arg Ile Arg Pro Gln Val Ser Glu Gly Phe 245 250 255Phe Ser Gln Asp Gly Pro Asn Val Gln Phe Glu Glu Leu Tyr Thr Phe 260 265270 Gly Gly Leu Asp Val Glu Ile Ile Gln Ile Glu Arg Ser Gln Leu Arg 275280 285 Glu Lys Ala Leu Gly His Tyr Lys Asp Ile Ala Lys Arg Leu Asn Asn290 295 300 Ile Asn Lys Thr Ile Pro Ser Ser Trp Ile Ser Asn Ile Asp LysTyr 305 310 315 320 Lys Lys Ile Phe Ser Glu Lys Tyr Asn Phe Asp Lys AspAsn Thr Gly 325 330 335 Asn Phe Val Val Asn Ile Asp Lys Phe Asn Ser LeuTyr Ser Asp Leu 340 345 350 Thr Asn Val Met Ser Glu Val Val Tyr Ser SerGln Tyr Asn Val Lys 355 360 365 Asn Arg Thr His Tyr Phe Ser Arg His TyrLeu Pro Val Phe Ala Asn 370 375 380 Ile Leu Asp Asp Asn Ile Tyr Thr IleArg Asp Gly Phe Asn Leu Thr 385 390 395 400 Asn Lys Gly Phe Asn Ile GluAsn Ser Gly Gln Asn Ile Glu Arg Asn 405 410 415 Pro Ala Leu Gln Lys LeuSer Ser Glu Ser Val Val Asp Leu Phe Thr 420 425 430 Lys Val Cys Leu ArgLeu Thr Lys Asn Ser Arg Asp Asp Ser Thr Cys 435 440 445 Ile Lys Val LysAsn Asn Arg Leu Pro Tyr Val Ala Asp Lys Asp Ser 450 455 460 Ile Ser GlnGlu Ile Phe Glu Asn Lys Ile Ile Thr Asp Glu Thr Asn 465 470 475 480 ValGln Asn Tyr Ser Asp Lys Phe Ser Leu Asp Glu Ser Ile Leu Asp 485 490 495Gly Gln Val Pro Ile Asn Pro Glu Ile Val Asp Pro Leu Leu Pro Asn 500 505510 Val Asn Met Glu Pro Leu Asn Leu Pro Gly Glu Glu Ile Val Phe Tyr 515520 525 Asp Asp Ile Thr Lys Tyr Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu530 535 540 Glu Ser Gln Lys Leu Ser Asn Asn Val Glu Asn Ile Thr Leu ThrThr 545 550 555 560 Ser Val Glu Glu Ala Leu Gly Tyr Ser Asn Lys Ile TyrThr Phe Leu 565 570 575 Pro Ser Leu Ala Glu Lys Val Asn Lys Gly Val GlnAla Gly Leu Phe 580 585 590 Leu Asn Trp Ala Asn Glu Val Val Glu Asp PheThr Thr Asn Ile Met 595 600 605 Lys Lys Asp Thr Leu Asp Lys Ile Ser AspVal Ser Val Ile Ile Pro 610 615 620 Tyr Ile Gly Pro Ala Leu Asn Ile GlyAsn Ser Ala Leu Arg Gly Asn 625 630 635 640 Phe Asn Gln Ala Phe Ala ThrAla Gly Val Ala Phe Leu Leu Glu Gly 645 650 655 Phe Pro Glu Phe Thr IlePro Ala Leu Gly Val Phe Thr Phe Tyr Ser 660 665 670 Ser Ile Gln Glu ArgGlu Lys Ile Ile Lys Thr Ile Glu Asn Cys Leu 675 680 685 Glu Gln Arg ValLys Arg Trp Lys Asp Ser Tyr Gln Trp Met Val Ser 690 695 700 Asn Trp LeuSer Arg Ile Thr Thr Gln Phe Asn His Ile Asn Tyr Gln 705 710 715 720 MetTyr Asp Ser Leu Ser Tyr Gln Ala Asp Ala Ile Lys Ala Lys Ile 725 730 735Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys 740 745750 Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu 755760 765 Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr770 775 780 Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn LysPhe 785 790 795 800 Asp Leu Arg Thr Lys Thr Glu Leu Ile Asn Leu Ile AspSer His Asn 805 810 815 Ile Ile Leu Val Gly Glu Val Asp 820 34 1283 DNAArtificial Sequence Synthetic polynucleotide sequence for the lightchain of of C. botulinum Type E, optimized for expression in E. coli. 34catatgccga aaatcaactc gttcaactac aacgacccgg tgaatgaccg cacaatcctg 60tacattaagc cgggcggttg ccaggagttc tacaagagct ttaacattat gaagaacatc 120tggatcatcc ctgaacgcaa tgtgatcggg acaacgccac aagatttcca ccctccgact 180tcgctcaaaa acggggactc ctcctactac gacccaaatt acttgcaaag cgatgaggag 240aaagatcggt tcctgaagat tgtgacaaag atcttcaacc gtattaacaa caatctctcg 300gggggcatcc tcctggagga attatccaag gcgaaccctt acctgggcaa cgacaacact 360ccagacaacc agttccacat tggcgacgcc tccgcggtgg agatcaagtt ctcgaatggc 420agtcaggaca tccttctccc taatgtcatt attatgggcg ccgagccgga cctttttgaa 480accaattcca gcaacatctc gctgcgcaac aactacatgc cgagcaatca cggctttggg 540tcgatcgcga tcgtgacttt ctcgccggag tactcctttc gcttcaacga caactccatg 600aacgagttca ttcaggaccc ggcgctcacc ctcatgcacg agctgatcca ctcgttacat 660ggcttgtacg gcgcgaaggg gatcacgacc aagtatacca ttacgcagaa acagaaccca 720cttatcacga acatccgtgg gacgaacatc gaggagttcc tcacgttcgg ggggaccgac 780ctgaacatta tcaccagcgc ccagtccaac gacatttaca cgaacctgct ggcagattac 840aaaaaaattg cctccaagct ctccaaggtc caggtatcga acccgttgct caatccttac 900aaggacgtct tcgaggctaa gtatgggctg gataaggatg cctcaggaat ctactctgtg 960aacatcaaca aattcaacga catcttcaag aagctgtaca gcttcaccga gtttgacctc 1020gccaccaagt tccaggtcaa atgtcggcaa acgtacattg gccagtataa atattttaag 1080ctgtcgaatc ttctcaacga ctctatctat aacatctccg aggggtacaa tattaacaac 1140ttaaaagtca acttccgagg gcagaacgca aatctcaacc cacggattat tactcctatt 1200acaggccgcg ggctcgtcaa gaagatcatc cgattttgca aaaacattgt cagcgttaaa 1260ggcatccgta agtaatagga tcc 1283 35 427 PRT Artificial SequenceRecombinant protein encoded by SEQ ID NO34 35 Met Pro Lys Ile Asn SerPhe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile Leu Tyr IleLys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn Ile Met LysAsn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr Thr Pro GlnAsp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser Ser Tyr TyrAsp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp Arg Phe LeuLys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn Leu Ser GlyGly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 Tyr Leu GlyAsn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125 Ala SerAla Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135 140 LeuPro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145 150 155160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser Asn His 165170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu Tyr Ser Phe180 185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln Asp Pro AlaLeu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His Gly Leu TyrGly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr Gln Lys GlnAsn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn Ile Glu GluPhe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile Thr Ser AlaGln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp Tyr Lys LysIle Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn Pro Leu LeuAsn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly Leu Asp LysAsp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn Lys Phe AsnAsp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe Asp Leu AlaThr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 Gly Gln TyrLys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365 Tyr AsnIle Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375 380 ArgGly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385 390 395400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn Ile Val 405410 415 Ser Val Lys Gly Ile Arg Lys Xaa Xaa Asp Xaa 420 425 36 2415 DNAArtificial Sequence Synthetic polynucleotide gene sequence for the lightchain with Hn segment of of C. botulinum Type E, optimized forexpression in E. coli. 36 catatgccga aaatcaactc gttcaactac aacgacccggtgaatgaccg cacaatcctg 60 tacattaagc cgggcggttg ccaggagttc tacaagagctttaacattat gaagaacatc 120 tggatcatcc ctgaacgcaa tgtgatcggg acaacgccacaagatttcca ccctccgact 180 tcgctcaaaa acggggactc ctcctactac gacccaaattacttgcaaag cgatgaggag 240 aaagatcggt tcctgaagat tgtgacaaag atcttcaaccgtattaacaa caatctctcg 300 gggggcatcc tcctggagga attatccaag gcgaacccttacctgggcaa cgacaacact 360 ccagacaacc agttccacat tggcgacgcc tccgcggtggagatcaagtt ctcgaatggc 420 agtcaggaca tccttctccc taatgtcatt attatgggcgccgagccgga cctttttgaa 480 accaattcca gcaacatctc gctgcgcaac aactacatgccgagcaatca cggctttggg 540 tcgatcgcga tcgtgacttt ctcgccggag tactcctttcgcttcaacga caactccatg 600 aacgagttca ttcaggaccc ggcgctcacc ctcatgcacgagctgatcca ctcgttacat 660 ggcttgtacg gcgcgaaggg gatcacgacc aagtataccattacgcagaa acagaaccca 720 cttatcacga acatccgtgg gacgaacatc gaggagttcctcacgttcgg ggggaccgac 780 ctgaacatta tcaccagcgc ccagtccaac gacatttacacgaacctgct ggcagattac 840 aaaaaaattg cctccaagct ctccaaggtc caggtatcgaacccgttgct caatccttac 900 aaggacgtct tcgaggctaa gtatgggctg gataaggatgcctcaggaat ctactctgtg 960 aacatcaaca aattcaacga catcttcaag aagctgtacagcttcaccga gtttgacctc 1020 gccaccaagt tccaggtcaa atgtcggcaa acgtacattggccagtataa atattttaag 1080 ctgtcgaatc ttctcaacga ctctatctat aacatctccgaggggtacaa tattaacaac 1140 ttaaaagtca acttccgagg gcagaacgca aatctcaacccacggattat tactcctatt 1200 acaggccgcg ggctcgtcaa gaagatcatc cgattttgcaaaaacattgt cagcgttaaa 1260 ggcatccgta agtccatctg catcgagatc aacaacggtgagctgttctt cgtggcttcc 1320 gagaacagtt acaacgatga caacatcaac actcctaaggagattgacga caccgtcact 1380 tctaacaaca actacgaaaa cgacctggac caggtcatcctaaacttcaa ctccgagtcc 1440 gcccctggtc tgtccgacga gaagctgaac ctgaccatccagaacgacgc ttacatccca 1500 aagtacgact ccaacggtac atccgatatc gagcagcatgacgttaacga gcttaacgtc 1560 ttcttctact tagacgctca gaaggtgccc gagggtgagaacaacgtcaa tctcacctct 1620 tcaattgaca cagccttgtt ggagcagcct aagatctacaccttcttctc ctccgagttc 1680 atcaacaacg tcaacaagcc tgtgcaggcc gcattgttcgtaagctggat tcagcaggtg 1740 ttagtagact tcactactga ggctaaccag aagtccactgttgacaagat cgctgacatc 1800 tccatcgtcg tcccatacat cggtctggct ctgaacatcggcaacgaggc acagaagggc 1860 aacttcaagg atgcccttga gttgttgggt gccggtattttgttggagtt cgaacccgag 1920 ctgctgatcc ctaccatcct ggtcttcacg atcaagtccttcctgggttc ctccgacaac 1980 aagaacaagg tcattaaggc catcaacaac gccctgaaggagcgtgacga gaagtggaag 2040 gaagtctatt ccttcatcgt ctcgaactgg atgaccaagatcaacaccca gttcaacaag 2100 cgaaaggagc agatgtacca ggctctgcag aaccaggtcaacgccatcaa gaccatcatc 2160 gagtccaagt acaactccta caccctggag gagaagaacgagcttaccaa caagtacgat 2220 atcaagcaga tcgagaacga gctgaaccag aaggtctccatcgccatgaa caacatcgac 2280 aggttcctga ccgagtcctc catctcctac ctgatgaagctcatcaacga ggtcaagatc 2340 aacaagctgc gagagtacga cgagaatgtc aagacgtacctgctgaacta catcatccag 2400 cacggatcca tcctg 2415 37 804 PRT ArtificialSequence Recombinant protein encoded by SEQ ID NO36 37 Met Pro Lys IleAsn Ser Phe Asn Tyr Asn Asp Pro Val Asn Asp Arg 1 5 10 15 Thr Ile LeuTyr Ile Lys Pro Gly Gly Cys Gln Glu Phe Tyr Lys Ser 20 25 30 Phe Asn IleMet Lys Asn Ile Trp Ile Ile Pro Glu Arg Asn Val Ile 35 40 45 Gly Thr ThrPro Gln Asp Phe His Pro Pro Thr Ser Leu Lys Asn Gly 50 55 60 Asp Ser SerTyr Tyr Asp Pro Asn Tyr Leu Gln Ser Asp Glu Glu Lys 65 70 75 80 Asp ArgPhe Leu Lys Ile Val Thr Lys Ile Phe Asn Arg Ile Asn Asn 85 90 95 Asn LeuSer Gly Gly Ile Leu Leu Glu Glu Leu Ser Lys Ala Asn Pro 100 105 110 TyrLeu Gly Asn Asp Asn Thr Pro Asp Asn Gln Phe His Ile Gly Asp 115 120 125Ala Ser Ala Val Glu Ile Lys Phe Ser Asn Gly Ser Gln Asp Ile Leu 130 135140 Leu Pro Asn Val Ile Ile Met Gly Ala Glu Pro Asp Leu Phe Glu Thr 145150 155 160 Asn Ser Ser Asn Ile Ser Leu Arg Asn Asn Tyr Met Pro Ser AsnHis 165 170 175 Gly Phe Gly Ser Ile Ala Ile Val Thr Phe Ser Pro Glu TyrSer Phe 180 185 190 Arg Phe Asn Asp Asn Ser Met Asn Glu Phe Ile Gln AspPro Ala Leu 195 200 205 Thr Leu Met His Glu Leu Ile His Ser Leu His GlyLeu Tyr Gly Ala 210 215 220 Lys Gly Ile Thr Thr Lys Tyr Thr Ile Thr GlnLys Gln Asn Pro Leu 225 230 235 240 Ile Thr Asn Ile Arg Gly Thr Asn IleGlu Glu Phe Leu Thr Phe Gly 245 250 255 Gly Thr Asp Leu Asn Ile Ile ThrSer Ala Gln Ser Asn Asp Ile Tyr 260 265 270 Thr Asn Leu Leu Ala Asp TyrLys Lys Ile Ala Ser Lys Leu Ser Lys 275 280 285 Val Gln Val Ser Asn ProLeu Leu Asn Pro Tyr Lys Asp Val Phe Glu 290 295 300 Ala Lys Tyr Gly LeuAsp Lys Asp Ala Ser Gly Ile Tyr Ser Val Asn 305 310 315 320 Ile Asn LysPhe Asn Asp Ile Phe Lys Lys Leu Tyr Ser Phe Thr Glu 325 330 335 Phe AspLeu Ala Thr Lys Phe Gln Val Lys Cys Arg Gln Thr Tyr Ile 340 345 350 GlyGln Tyr Lys Tyr Phe Lys Leu Ser Asn Leu Leu Asn Asp Ser Ile 355 360 365Tyr Asn Ile Ser Glu Gly Tyr Asn Ile Asn Asn Leu Lys Val Asn Phe 370 375380 Arg Gly Gln Asn Ala Asn Leu Asn Pro Arg Ile Ile Thr Pro Ile Thr 385390 395 400 Gly Arg Gly Leu Val Lys Lys Ile Ile Arg Phe Cys Lys Asn IleVal 405 410 415 Ser Val Lys Gly Ile Arg Lys Ser Ile Cys Ile Glu Ile AsnAsn Gly 420 425 430 Glu Leu Phe Phe Val Ala Ser Glu Asn Ser Tyr Asn AspAsp Asn Ile 435 440 445 Asn Thr Pro Lys Glu Ile Asp Asp Thr Val Thr SerAsn Asn Asn Tyr 450 455 460 Glu Asn Asp Leu Asp Gln Val Ile Leu Asn PheAsn Ser Glu Ser Ala 465 470 475 480 Pro Gly Leu Ser Asp Glu Lys Leu AsnLeu Thr Ile Gln Asn Asp Ala 485 490 495 Tyr Ile Pro Lys Tyr Asp Ser AsnGly Thr Ser Asp Ile Glu Gln His 500 505 510 Asp Val Asn Glu Leu Asn ValPhe Phe Tyr Leu Asp Ala Gln Lys Val 515 520 525 Pro Glu Gly Glu Asn AsnVal Asn Leu Thr Ser Ser Ile Asp Thr Ala 530 535 540 Leu Leu Glu Gln ProLys Ile Tyr Thr Phe Phe Ser Ser Glu Phe Ile 545 550 555 560 Asn Asn ValAsn Lys Pro Val Gln Ala Ala Leu Phe Val Ser Trp Ile 565 570 575 Gln GlnVal Leu Val Asp Phe Thr Thr Glu Ala Asn Gln Lys Ser Thr 580 585 590 ValAsp Lys Ile Ala Asp Ile Ser Ile Val Val Pro Tyr Ile Gly Leu 595 600 605Ala Leu Asn Ile Gly Asn Glu Ala Gln Lys Gly Asn Phe Lys Asp Ala 610 615620 Leu Glu Leu Leu Gly Ala Gly Ile Leu Leu Glu Phe Glu Pro Glu Leu 625630 635 640 Leu Ile Pro Thr Ile Leu Val Phe Thr Ile Lys Ser Phe Leu GlySer 645 650 655 Ser Asp Asn Lys Asn Lys Val Ile Lys Ala Ile Asn Asn AlaLeu Lys 660 665 670 Glu Arg Asp Glu Lys Trp Lys Glu Val Tyr Ser Phe IleVal Ser Asn 675 680 685 Trp Met Thr Lys Ile Asn Thr Gln Phe Asn Lys ArgLys Glu Gln Met 690 695 700 Tyr Gln Ala Leu Gln Asn Gln Val Asn Ala IleLys Thr Ile Ile Glu 705 710 715 720 Ser Lys Tyr Asn Ser Tyr Thr Leu GluGlu Lys Asn Glu Leu Thr Asn 725 730 735 Lys Tyr Asp Ile Lys Gln Ile GluAsn Glu Leu Asn Gln Lys Val Ser 740 745 750 Ile Ala Met Asn Asn Ile AspArg Phe Leu Thr Glu Ser Ser Ile Ser 755 760 765 Tyr Leu Met Lys Leu IleAsn Glu Val Lys Ile Asn Lys Leu Arg Glu 770 775 780 Tyr Asp Glu Asn ValLys Thr Tyr Leu Leu Asn Tyr Ile Ile Gln His 785 790 795 800 Gly Ser IleLeu 38 1334 DNA Artificial Sequence Synthetic polynucleotide sequencefor the light chain of of C. botulinum Type F, optimized for expressionin E. coli. 38 catatgccgg ttgtcatcaa ttcttttaac tacaacgacc cggtgaacgacgacacgatt 60 ctgtacatgc aaatccctta cgaggagaag tctaaaaagt attataaggcgttcgagatc 120 atgcgcaacg tgtggatcat cccggaacgc aacactattg ggacagacccgtcggacttc 180 gatccgcctg cgtcgcttga aaacggctca tcagcatact atgacccaaattatttgact 240 acggacgcgg aaaaggaccg ttatctcaag accacaatca agctcttcaagcgtattaac 300 tccaacccgg cgggcgaggt attgcttcag gagatttcct acgccaagccttacctcggc 360 aatgagcata ctcctatcaa cgagttccac cctgtgaccc gaaccacgtctgtaaacatt 420 aagagttcga cgaatgtaaa gtcgtcaatt attctcaacc tcttggtccttggcgcgggg 480 ccggacatct tcgagaactc ttcctacccg gttcgcaagc tcatggacagtgggggggtc 540 tatgacccga gcaacgacgg gttcggttcc atcaatatcg tgaccttctcacctgagtac 600 gagtatacat ttaacgacat cagcggcggc tacaacagta gcaccgagtcctttatcgcc 660 gacccggcca tcagcctcgc tcacgagctc atccacgccc tgcacgggctgtacggggcc 720 cggggcgtta catataagga gaccatcaaa gtgaagcagg cgccactcatgattgccgaa 780 aagccaatcc gattggagga gttcctgaca ttcgggggcc aggacctgaatattatcact 840 agtgcaatga aggagaagat ttataacaac ctgctcgcga actatgagaagatcgccact 900 cgcttatccc gggtgaactc cgccccaccg gagtatgaca ttaacgagtataaagactac 960 ttccagtgga agtatggact ggataaaaac gcggacgggt cttacaccgtgaacgagaac 1020 aaattcaacg agatctacaa gaagctctac agcttcacgg agatcgacctcgcgaacaag 1080 ttcaaggtga agtgccggaa cacgtatttc atcaagtacg gcttcttaaaggtgccaaac 1140 ctgttagacg acgacattta taccgtatcg gagggcttca atattggtaatctggccgtg 1200 aacaatcgcg gccagaatat taaacttaac ccgaaaatta tcgactcgatcccagacaag 1260 gggttagttg agaagatcgt caagttctgc aagtcggtca tccctcgcaaggggacgaag 1320 aattaatagg atcc 1334 39 443 PRT Artificial SequenceRecombinant protein encoded by SEQ ID NO38 39 Met Pro Val Val Ile AsnSer Phe Asn Tyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu TyrMet Gln Ile Pro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala PheGlu Ile Met Arg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn Thr Ile GlyThr Asp Pro Ser Asp Phe Asp Pro Pro Ala Ser 50 55 60 Leu Glu Asn Gly SerSer Ala Tyr Tyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 Asp Ala Glu LysAsp Arg Tyr Leu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95 Arg Ile Asn SerAsn Pro Ala Gly Glu Val Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala LysPro Tyr Leu Gly Asn Glu His Thr Pro Ile Asn Glu Phe 115 120 125 His ProVal Thr Arg Thr Thr Ser Val Asn Ile Lys Ser Ser Thr Asn 130 135 140 ValLys Ser Ser Ile Ile Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155160 Asp Ile Phe Glu Asn Ser Ser Tyr Pro Val Arg Lys Leu Met Asp Ser 165170 175 Gly Gly Val Tyr Asp Pro Ser Asn Asp Gly Phe Gly Ser Ile Asn Ile180 185 190 Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile SerGly 195 200 205 Gly Tyr Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro AlaIle Ser 210 215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu TyrGly Ala Arg 225 230 235 240 Gly Val Thr Tyr Lys Glu Thr Ile Lys Val LysGln Ala Pro Leu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu GluPhe Leu Thr Phe Gly Gly 260 265 270 Gln Asp Leu Asn Ile Ile Thr Ser AlaMet Lys Glu Lys Ile Tyr Asn 275 280 285 Asn Leu Leu Ala Asn Tyr Glu LysIle Ala Thr Arg Leu Ser Arg Val 290 295 300 Asn Ser Ala Pro Pro Glu TyrAsp Ile Asn Glu Tyr Lys Asp Tyr Phe 305 310 315 320 Gln Trp Lys Tyr GlyLeu Asp Lys Asn Ala Asp Gly Ser Tyr Thr Val 325 330 335 Asn Glu Asn LysPhe Asn Glu Ile Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ile AspLeu Ala Asn Lys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe IleLys Tyr Gly Phe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 IleTyr Thr Val Ser Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395400 Asn Arg Gly Gln Asn Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405410 415 Pro Asp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val420 425 430 Ile Pro Arg Lys Gly Thr Lys Asn Xaa Xaa Asp 435 440 40 2577DNA Artificial Sequence Synthetic polynucleotide sequence for the lightchain with Hn segment of of C. botulinum Type F, optimized forexpression in E. coli. 40 catatgccgg ttgtcatcaa ttcttttaac tacaacgacccggtgaacga cgacacgatt 60 ctgtacatgc aaatccctta cgaggagaag tctaaaaagtattataaggc gttcgagatc 120 atgcgcaacg tgtggatcat cccggaacgc aacactattgggacagaccc gtcggacttc 180 gatccgcctg cgtcgcttga aaacggctca tcagcatactatgacccaaa ttatttgact 240 acggacgcgg aaaaggaccg ttatctcaag accacaatcaagctcttcaa gcgtattaac 300 tccaacccgg cgggcgaggt attgcttcag gagatttcctacgccaagcc ttacctcggc 360 aatgagcata ctcctatcaa cgagttccac cctgtgacccgaaccacgtc tgtaaacatt 420 aagagttcga cgaatgtaaa gtcgtcaatt attctcaacctcttggtcct tggcgcgggg 480 ccggacatct tcgagaactc ttcctacccg gttcgcaagctcatggacag tgggggggtc 540 tatgacccga gcaacgacgg gttcggttcc atcaatatcgtgaccttctc acctgagtac 600 gagtatacat ttaacgacat cagcggcggc tacaacagtagcaccgagtc ctttatcgcc 660 gacccggcca tcagcctcgc tcacgagctc atccacgccctgcacgggct gtacggggcc 720 cggggcgtta catataagga gaccatcaaa gtgaagcaggcgccactcat gattgccgaa 780 aagccaatcc gattggagga gttcctgaca ttcgggggccaggacctgaa tattatcact 840 agtgcaatga aggagaagat ttataacaac ctgctcgcgaactatgagaa gatcgccact 900 cgcttatccc gggtgaactc cgccccaccg gagtatgacattaacgagta taaagactac 960 ttccagtgga agtatggact ggataaaaac gcggacgggtcttacaccgt gaacgagaac 1020 aaattcaacg agatctacaa gaagctctac agcttcacggagatcgacct cgcgaacaag 1080 ttcaaggtga agtgccggaa cacgtatttc atcaagtacggcttcttaaa ggtgccaaac 1140 ctgttagacg acgacattta taccgtatcg gagggcttcaatattggtaa tctggccgtg 1200 aacaatcgcg gccagaatat taaacttaac ccgaaaattatcgactcgat cccagacaag 1260 gggttagttg agaagatcgt caagttctgc aagtcggtcatccctcgcaa ggggacgaag 1320 aattgcaagt ccgtcatccc acgtaagggt accaaggccccaccacgtct gtgtattaga 1380 gtcaacaact cagaattatt ctttgtcgct tccgagtcaagctacaacga gaacgatatt 1440 aacacaccta aagagattga cgatactacc aacctaaacaacaactaccg gaacaacttg 1500 gatgaggtta ttttggatta caactcacag accatccctcaaatttccaa ccgtacctta 1560 aacactcttg tccaagacaa ctcctacgtt ccaagatacgattctaacgg tacctcagag 1620 atcgaggagt atgatgttgt tgactttaac gtctttttctatttgcatgc ccagaaggtg 1680 ccagaaggtg aaaccaacat ctcattgact tcttccattgataccgcctt gttggaagag 1740 tccaaggata tcttcttttc ttcggagttt atcgatactatcaacaagcc tgtcaacgcc 1800 gctctgttca ttgattggat tagcaaggtc atcagagattttaccactga agctactcaa 1860 aagtccactg ttgataagat tgctgacatc tctttgattgtcccctatgt cggtcttgct 1920 ttgaacatca ttattgaggc agaaaagggt aactttgaggaggcttttga attgttggga 1980 gttggtattt tgttggagtt tgttccagaa cttaccattcctgtcatttt agtttttacg 2040 atcaagtcct acatcgattc atacgagaac aagaataaagcaattaaagc tattaacaac 2100 tccttgatcg aaagagaggc taagtggaag gaaatctactcatggattgt atcaaactgg 2160 cttactagaa ttaacactca atttaacaag agaaaggagcaaatgtacca ggctctgcaa 2220 aaccaagtcg atgctatcaa gactgcaatt gaatacaagtacaacaacta tacttccgat 2280 gagaagaaca gacttgaatc tgaatacaat atcaacaacattgaagaaga gttgaacaag 2340 aaagtttctt tggctatgaa gaatatcgaa agatttatgaccgaatcctc tatctcttac 2400 ttgatgaagt tgatcaatga ggccaaggtt ggtaagttgaagaagtacga taaccacgtt 2460 aagagcgatc tgctgaacta cattctcgac cacagatcaatcctgggaga gcagacaaac 2520 gagctgagtg atttggttac ttccactttg aactcctccattccatttga gctttct 2577 41 858 PRT Artificial Sequence Recombinantprotein encoded by SEQ ID NO40 41 Met Pro Val Val Ile Asn Ser Phe AsnTyr Asn Asp Pro Val Asn Asp 1 5 10 15 Asp Thr Ile Leu Tyr Met Gln IlePro Tyr Glu Glu Lys Ser Lys Lys 20 25 30 Tyr Tyr Lys Ala Phe Glu Ile MetArg Asn Val Trp Ile Ile Pro Glu 35 40 45 Arg Asn Thr Ile Gly Thr Asp ProSer Asp Phe Asp Pro Pro Ala Ser 50 55 60 Leu Glu Asn Gly Ser Ser Ala TyrTyr Asp Pro Asn Tyr Leu Thr Thr 65 70 75 80 Asp Ala Glu Lys Asp Arg TyrLeu Lys Thr Thr Ile Lys Leu Phe Lys 85 90 95 Arg Ile Asn Ser Asn Pro AlaGly Glu Val Leu Leu Gln Glu Ile Ser 100 105 110 Tyr Ala Lys Pro Tyr LeuGly Asn Glu His Thr Pro Ile Asn Glu Phe 115 120 125 His Pro Val Thr ArgThr Thr Ser Val Asn Ile Lys Ser Ser Thr Asn 130 135 140 Val Lys Ser SerIle Ile Leu Asn Leu Leu Val Leu Gly Ala Gly Pro 145 150 155 160 Asp IlePhe Glu Asn Ser Ser Tyr Pro Val Arg Lys Leu Met Asp Ser 165 170 175 GlyGly Val Tyr Asp Pro Ser Asn Asp Gly Phe Gly Ser Ile Asn Ile 180 185 190Val Thr Phe Ser Pro Glu Tyr Glu Tyr Thr Phe Asn Asp Ile Ser Gly 195 200205 Gly Tyr Asn Ser Ser Thr Glu Ser Phe Ile Ala Asp Pro Ala Ile Ser 210215 220 Leu Ala His Glu Leu Ile His Ala Leu His Gly Leu Tyr Gly Ala Arg225 230 235 240 Gly Val Thr Tyr Lys Glu Thr Ile Lys Val Lys Gln Ala ProLeu Met 245 250 255 Ile Ala Glu Lys Pro Ile Arg Leu Glu Glu Phe Leu ThrPhe Gly Gly 260 265 270 Gln Asp Leu Asn Ile Ile Thr Ser Ala Met Lys GluLys Ile Tyr Asn 275 280 285 Asn Leu Leu Ala Asn Tyr Glu Lys Ile Ala ThrArg Leu Ser Arg Val 290 295 300 Asn Ser Ala Pro Pro Glu Tyr Asp Ile AsnGlu Tyr Lys Asp Tyr Phe 305 310 315 320 Gln Trp Lys Tyr Gly Leu Asp LysAsn Ala Asp Gly Ser Tyr Thr Val 325 330 335 Asn Glu Asn Lys Phe Asn GluIle Tyr Lys Lys Leu Tyr Ser Phe Thr 340 345 350 Glu Ile Asp Leu Ala AsnLys Phe Lys Val Lys Cys Arg Asn Thr Tyr 355 360 365 Phe Ile Lys Tyr GlyPhe Leu Lys Val Pro Asn Leu Leu Asp Asp Asp 370 375 380 Ile Tyr Thr ValSer Glu Gly Phe Asn Ile Gly Asn Leu Ala Val Asn 385 390 395 400 Asn ArgGly Gln Asn Ile Lys Leu Asn Pro Lys Ile Ile Asp Ser Ile 405 410 415 ProAsp Lys Gly Leu Val Glu Lys Ile Val Lys Phe Cys Lys Ser Val 420 425 430Ile Pro Arg Lys Gly Thr Lys Asn Cys Lys Ser Val Ile Pro Arg Lys 435 440445 Gly Thr Lys Ala Pro Pro Arg Leu Cys Ile Arg Val Asn Asn Ser Glu 450455 460 Leu Phe Phe Val Ala Ser Glu Ser Ser Tyr Asn Glu Asn Asp Ile Asn465 470 475 480 Thr Pro Lys Glu Ile Asp Asp Thr Thr Asn Leu Asn Asn AsnTyr Arg 485 490 495 Asn Asn Leu Asp Glu Val Ile Leu Asp Tyr Asn Ser GlnThr Ile Pro 500 505 510 Gln Ile Ser Asn Arg Thr Leu Asn Thr Leu Val GlnAsp Asn Ser Tyr 515 520 525 Val Pro Arg Tyr Asp Ser Asn Gly Thr Ser GluIle Glu Glu Tyr Asp 530 535 540 Val Val Asp Phe Asn Val Phe Phe Tyr LeuHis Ala Gln Lys Val Pro 545 550 555 560 Glu Gly Glu Thr Asn Ile Ser LeuThr Ser Ser Ile Asp Thr Ala Leu 565 570 575 Leu Glu Glu Ser Lys Asp IlePhe Phe Ser Ser Glu Phe Ile Asp Thr 580 585 590 Ile Asn Lys Pro Val AsnAla Ala Leu Phe Ile Asp Trp Ile Ser Lys 595 600 605 Val Ile Arg Asp PheThr Thr Glu Ala Thr Gln Lys Ser Thr Val Asp 610 615 620 Lys Ile Ala AspIle Ser Leu Ile Val Pro Tyr Val Gly Leu Ala Leu 625 630 635 640 Asn IleIle Ile Glu Ala Glu Lys Gly Asn Phe Glu Glu Ala Phe Glu 645 650 655 LeuLeu Gly Val Gly Ile Leu Leu Glu Phe Val Pro Glu Leu Thr Ile 660 665 670Pro Val Ile Leu Val Phe Thr Ile Lys Ser Tyr Ile Asp Ser Tyr Glu 675 680685 Asn Lys Asn Lys Ala Ile Lys Ala Ile Asn Asn Ser Leu Ile Glu Arg 690695 700 Glu Ala Lys Trp Lys Glu Ile Tyr Ser Trp Ile Val Ser Asn Trp Leu705 710 715 720 Thr Arg Ile Asn Thr Gln Phe Asn Lys Arg Lys Glu Gln MetTyr Gln 725 730 735 Ala Leu Gln Asn Gln Val Asp Ala Ile Lys Thr Ala IleGlu Tyr Lys 740 745 750 Tyr Asn Asn Tyr Thr Ser Asp Glu Lys Asn Arg LeuGlu Ser Glu Tyr 755 760 765 Asn Ile Asn Asn Ile Glu Glu Glu Leu Asn LysLys Val Ser Leu Ala 770 775 780 Met Lys Asn Ile Glu Arg Phe Met Thr GluSer Ser Ile Ser Tyr Leu 785 790 795 800 Met Lys Leu Ile Asn Glu Ala LysVal Gly Lys Leu Lys Lys Tyr Asp 805 810 815 Asn His Val Lys Ser Asp LeuLeu Asn Tyr Ile Leu Asp His Arg Ser 820 825 830 Ile Leu Gly Glu Gln ThrAsn Glu Leu Ser Asp Leu Val Thr Ser Thr 835 840 845 Leu Asn Ser Ser IlePro Phe Glu Leu Ser 850 855 42 1337 DNA Artificial Sequence Syntheticpolynucleotide sequence for the light chain of of C. botulinum Type G,optimized for expression in E. coli. 42 catatgccgg tcaatattaa gaacttcaattacaacgacc cgatcaataa tgacgatatc 60 attatgatgg agcctttcaa cgacccaggtccaggcacgt attacaaggc ttttcggatc 120 atcgaccgca tttggatcgt cccggagcgcttcacgtacg gcttccaacc tgaccagttc 180 aatgcaagca caggggtttt cagcaaggacgtctacgagt actatgaccc aacttacctg 240 aagactgacg cggagaagga caaattcctgaagacgatga tcaagttgtt caaccgcatt 300 aactccaagc cgtccggcca gcgactgcttgatatgattg tggacgccat cccttacctc 360 ggaaacgcct ctacgccacc ggacaagttcgcggcaaacg ttgcaaacgt gtccatcaac 420 aagaaaatta ttcagccggg ggccgaggaccagattaagg gacttatgac taatctgatc 480 atcttcgggc cggggcctgt actctcggacaacttcacgg acagcatgat tatgaacggc 540 cattcaccga tctcagaagg attcggggcacgtatgatga tccggttctg cccgagttgc 600 ctcaacgtct tcaacaacgt ccaggaaaataaggatacat cgatcttctc ccgccgtgcc 660 tacttcgcgg acccagcgtt aacccttatgcacgagttaa tccacgtatt gcacggcctc 720 tacggcatta agatctcgaa cttacctattaccccaaaca cgaaagagtt cttcatgcaa 780 cacagcgacc cggttcaggc cgaggaattatacaccttcg gcgggcacga cccaagtgtt 840 atctcaccgt ctaccgatat gaatatctacaacaaggccc tgcaaaactt ccaggacatc 900 gcaaaccggc ttaacattgt ctcatcggcacaggggtctg gtatcgacat ctccctgtat 960 aagcagatct acaagaataa gtacgacttcgtagaagacc cgaacggcaa gtactcggtg 1020 gacaaggaca agtttgacaa actctacaaagctctcatgt tcggtttcac agagacaaat 1080 cttgccggag agtacgggat caagacgcggtactcgtatt tttccgagta cctgccgcct 1140 attaagacgg agaagttgct cgataacaccatttacactc agaatgaggg gttcaacatc 1200 gcctctaaga atctcaagac cgagttcaatggtcagaaca aggcggtgaa caaagaggcg 1260 tatgaggaga ttagtctgga acacttggtgatctaccgaa ttgcgatgtg taagcctgtg 1320 atgtactaat aggatcc 1337 43 444 PRTArtificial Sequence Recombinant protein encoded by SEQ ID NO42 43 MetPro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn 1 5 10 15Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro Gly Thr 20 25 30Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val Pro Glu 35 40 45Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser Thr Gly 50 55 60Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr Leu Lys 65 70 7580 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile Lys Leu Phe 85 9095 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu Asp Met Ile 100105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr Pro Pro Asp Lys115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn Lys Lys Ile IleGln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu Met Thr Asn LeuIle Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser Asp Asn Phe ThrAsp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro Ile Ser Glu Gly PheGly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro Ser Cys Leu Asn ValPhe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp Thr Ser Ile Phe Ser ArgArg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala Leu Thr Leu Met His Glu LeuIle His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile Lys Ile Ser AsnLeu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe Met Gln His SerAsp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 Gly Gly His AspPro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285 Tyr Asn LysAla Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295 300 Ile ValSer Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys 305 310 315 320Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn Gly Lys 325 330335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys Ala Leu Met 340345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr Gly Ile Lys Thr355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro Ile Lys Thr GluLys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn Glu Gly Phe AsnIle Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe Asn Gly Gln AsnLys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu Ile Ser Leu Glu HisLeu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys Pro Val Met Tyr XaaXaa Asp 435 440 44 2547 DNA Artificial Sequence Synthetic polynucleotidesequence for the light chain with Hn segment of of C. botulinum Type G,optimized for expression in E. coli. 44 catatgccgg tcaatattaa gaacttcaattacaacgacc cgatcaataa tgacgatatc 60 attatgatgg agcctttcaa cgacccaggtccaggcacgt attacaaggc ttttcggatc 120 atcgaccgca tttggatcgt cccggagcgcttcacgtacg gcttccaacc tgaccagttc 180 aatgcaagca caggggtttt cagcaaggacgtctacgagt actatgaccc aacttacctg 240 aagactgacg cggagaagga caaattcctgaagacgatga tcaagttgtt caaccgcatt 300 aactccaagc cgtccggcca gcgactgcttgatatgattg tggacgccat cccttacctc 360 ggaaacgcct ctacgccacc ggacaagttcgcggcaaacg ttgcaaacgt gtccatcaac 420 aagaaaatta ttcagccggg ggccgaggaccagattaagg gacttatgac taatctgatc 480 atcttcgggc cggggcctgt actctcggacaacttcacgg acagcatgat tatgaacggc 540 cattcaccga tctcagaagg attcggggcacgtatgatga tccggttctg cccgagttgc 600 ctcaacgtct tcaacaacgt ccaggaaaataaggatacat cgatcttctc ccgccgtgcc 660 tacttcgcgg acccagcgtt aacccttatgcacgagttaa tccacgtatt gcacggcctc 720 tacggcatta agatctcgaa cttacctattaccccaaaca cgaaagagtt cttcatgcaa 780 cacagcgacc cggttcaggc cgaggaattatacaccttcg gcgggcacga cccaagtgtt 840 atctcaccgt ctaccgatat gaatatctacaacaaggccc tgcaaaactt ccaggacatc 900 gcaaaccggc ttaacattgt ctcatcggcacaggggtctg gtatcgacat ctccctgtat 960 aagcagatct acaagaataa gtacgacttcgtagaagacc cgaacggcaa gtactcggtg 1020 gacaaggaca agtttgacaa actctacaaagctctcatgt tcggtttcac agagacaaat 1080 cttgccggag agtacgggat caagacgcggtactcgtatt tttccgagta cctgccgcct 1140 attaagacgg agaagttgct cgataacaccatttacactc agaatgaggg gttcaacatc 1200 gcctctaaga atctcaagac cgagttcaatggtcagaaca aggcggtgaa caaagaggcg 1260 tatgaggaga ttagtctgga acacttggtgatctaccgaa ttgcgatgtg taagcctgtg 1320 atgtacaaga acaccggtaa gtccgagcagtgtatcatcg tcaacaacga ggacttgttc 1380 ttcatcgcca acaaggactc cttctccaaggacttggcca aggctgagac catcgcctac 1440 aacacccaga acaacaccat cgagaacaacttctccatcg accagctgat cttggacaac 1500 gacctgtcct ccggtatcga cctgccaaacgagaacaccg agccattcac caacttcgac 1560 gacatcgaca tcccagtcta catcaagcagtccgccctga agaagatctt cgtcgacggt 1620 gactccttgt tcgagtacct gcacgcccagaccttcccat ccaacatcga gaaccagttg 1680 accaactccc tgaacgacgc tttgagaaacaacaacaagg tctacacctt cttctccact 1740 aacttggtcg agaaggccaa cactgtcgtcggtgcctcct tgttcgtcaa ctgggtcaag 1800 ggtgtcatcg acgacttcac ctccgagtccacccaaaagt ccaccatcga caaggtctcc 1860 gacgtctcca tcatcatccc atacatcggtccagccctga acgtcggtaa cgagaccgct 1920 aaggagaact tcaagaacgc cttcgagatcggtggtgccg ccatcctgat ggagttcatc 1980 ccagagttga tcgtcccaat cgtcggtttcttcaccttgg agtcctacgt cggtaacaag 2040 ggtcacatca tcatgaccat ctccaacgccctgaagaaga gagaccagaa gtggaccgac 2100 atgtacggtt tgatcgtctc ccagtggttgtccaccgtca acacccagtt ctacaccatc 2160 aaggagagaa tgtacaacgc cttgaacaaccagtcccagg ccatcgagaa gatcatcgag 2220 gaccagtaca accgttactc cgaggaggacaagatgaaca tcaacatcga cttcaacgac 2280 atcgacttca agctgaacca gtccatcaacctggccatca acaacatcga cgacttcatc 2340 aaccagtgtt ccatctccta cctgatgaaccgtatgatcc cactggccgt caagaagttg 2400 aaggacttcg acgacaacct gaagcgtgacctgctggagt acatcgacac caacgagttg 2460 tacctgctgg acgaggtcaa catcttgaagtccaaggtca acagacactt gaaggactcc 2520 atcccattcg acttgtcctt gtacacc 254745 848 PRT Artificial Sequence Recombinant protein encoded by SEQ IDNO44 45 Met Pro Val Asn Ile Lys Asn Phe Asn Tyr Asn Asp Pro Ile Asn Asn1 5 10 15 Asp Asp Ile Ile Met Met Glu Pro Phe Asn Asp Pro Gly Pro GlyThr 20 25 30 Tyr Tyr Lys Ala Phe Arg Ile Ile Asp Arg Ile Trp Ile Val ProGlu 35 40 45 Arg Phe Thr Tyr Gly Phe Gln Pro Asp Gln Phe Asn Ala Ser ThrGly 50 55 60 Val Phe Ser Lys Asp Val Tyr Glu Tyr Tyr Asp Pro Thr Tyr LeuLys 65 70 75 80 Thr Asp Ala Glu Lys Asp Lys Phe Leu Lys Thr Met Ile LysLeu Phe 85 90 95 Asn Arg Ile Asn Ser Lys Pro Ser Gly Gln Arg Leu Leu AspMet Ile 100 105 110 Val Asp Ala Ile Pro Tyr Leu Gly Asn Ala Ser Thr ProPro Asp Lys 115 120 125 Phe Ala Ala Asn Val Ala Asn Val Ser Ile Asn LysLys Ile Ile Gln 130 135 140 Pro Gly Ala Glu Asp Gln Ile Lys Gly Leu MetThr Asn Leu Ile Ile 145 150 155 160 Phe Gly Pro Gly Pro Val Leu Ser AspAsn Phe Thr Asp Ser Met Ile 165 170 175 Met Asn Gly His Ser Pro Ile SerGlu Gly Phe Gly Ala Arg Met Met 180 185 190 Ile Arg Phe Cys Pro Ser CysLeu Asn Val Phe Asn Asn Val Gln Glu 195 200 205 Asn Lys Asp Thr Ser IlePhe Ser Arg Arg Ala Tyr Phe Ala Asp Pro 210 215 220 Ala Leu Thr Leu MetHis Glu Leu Ile His Val Leu His Gly Leu Tyr 225 230 235 240 Gly Ile LysIle Ser Asn Leu Pro Ile Thr Pro Asn Thr Lys Glu Phe 245 250 255 Phe MetGln His Ser Asp Pro Val Gln Ala Glu Glu Leu Tyr Thr Phe 260 265 270 GlyGly His Asp Pro Ser Val Ile Ser Pro Ser Thr Asp Met Asn Ile 275 280 285Tyr Asn Lys Ala Leu Gln Asn Phe Gln Asp Ile Ala Asn Arg Leu Asn 290 295300 Ile Val Ser Ser Ala Gln Gly Ser Gly Ile Asp Ile Ser Leu Tyr Lys 305310 315 320 Gln Ile Tyr Lys Asn Lys Tyr Asp Phe Val Glu Asp Pro Asn GlyLys 325 330 335 Tyr Ser Val Asp Lys Asp Lys Phe Asp Lys Leu Tyr Lys AlaLeu Met 340 345 350 Phe Gly Phe Thr Glu Thr Asn Leu Ala Gly Glu Tyr GlyIle Lys Thr 355 360 365 Arg Tyr Ser Tyr Phe Ser Glu Tyr Leu Pro Pro IleLys Thr Glu Lys 370 375 380 Leu Leu Asp Asn Thr Ile Tyr Thr Gln Asn GluGly Phe Asn Ile Ala 385 390 395 400 Ser Lys Asn Leu Lys Thr Glu Phe AsnGly Gln Asn Lys Ala Val Asn 405 410 415 Lys Glu Ala Tyr Glu Glu Ile SerLeu Glu His Leu Val Ile Tyr Arg 420 425 430 Ile Ala Met Cys Lys Pro ValMet Tyr Lys Asn Thr Gly Lys Ser Glu 435 440 445 Gln Cys Ile Ile Val AsnAsn Glu Asp Leu Phe Phe Ile Ala Asn Lys 450 455 460 Asp Ser Phe Ser LysAsp Leu Ala Lys Ala Glu Thr Ile Ala Tyr Asn 465 470 475 480 Thr Gln AsnAsn Thr Ile Glu Asn Asn Phe Ser Ile Asp Gln Leu Ile 485 490 495 Leu AspAsn Asp Leu Ser Ser Gly Ile Asp Leu Pro Asn Glu Asn Thr 500 505 510 GluPro Phe Thr Asn Phe Asp Asp Ile Asp Ile Pro Val Tyr Ile Lys 515 520 525Gln Ser Ala Leu Lys Lys Ile Phe Val Asp Gly Asp Ser Leu Phe Glu 530 535540 Tyr Leu His Ala Gln Thr Phe Pro Ser Asn Ile Glu Asn Gln Leu Thr 545550 555 560 Asn Ser Leu Asn Asp Ala Leu Arg Asn Asn Asn Lys Val Tyr ThrPhe 565 570 575 Phe Ser Thr Asn Leu Val Glu Lys Ala Asn Thr Val Val GlyAla Ser 580 585 590 Leu Phe Val Asn Trp Val Lys Gly Val Ile Asp Asp PheThr Ser Glu 595 600 605 Ser Thr Gln Lys Ser Thr Ile Asp Lys Val Ser AspVal Ser Ile Ile 610 615 620 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Val GlyAsn Glu Thr Ala Lys 625 630 635 640 Glu Asn Phe Lys Asn Ala Phe Glu IleGly Gly Ala Ala Ile Leu Met 645 650 655 Glu Phe Ile Pro Glu Leu Ile ValPro Ile Val Gly Phe Phe Thr Leu 660 665 670 Glu Ser Tyr Val Gly Asn LysGly His Ile Ile Met Thr Ile Ser Asn 675 680 685 Ala Leu Lys Lys Arg AspGln Lys Trp Thr Asp Met Tyr Gly Leu Ile 690 695 700 Val Ser Gln Trp LeuSer Thr Val Asn Thr Gln Phe Tyr Thr Ile Lys 705 710 715 720 Glu Arg MetTyr Asn Ala Leu Asn Asn Gln Ser Gln Ala Ile Glu Lys 725 730 735 Ile IleGlu Asp Gln Tyr Asn Arg Tyr Ser Glu Glu Asp Lys Met Asn 740 745 750 IleAsn Ile Asp Phe Asn Asp Ile Asp Phe Lys Leu Asn Gln Ser Ile 755 760 765Asn Leu Ala Ile Asn Asn Ile Asp Asp Phe Ile Asn Gln Cys Ser Ile 770 775780 Ser Tyr Leu Met Asn Arg Met Ile Pro Leu Ala Val Lys Lys Leu Lys 785790 795 800 Asp Phe Asp Asp Asn Leu Lys Arg Asp Leu Leu Glu Tyr Ile AspThr 805 810 815 Asn Glu Leu Tyr Leu Leu Asp Glu Val Asn Ile Leu Lys SerLys Val 820 825 830 Asn Arg His Leu Lys Asp Ser Ile Pro Phe Asp Leu SerLeu Tyr Thr 835 840 845 46 7 PRT Artificial Sequence Synthetic peptide;competative inhibitor of Zn protease 46 Cys Arg Ala Thr Lys Met Leu 1 547 449 PRT Artificial Sequence Synthetic botulinum neurotoxin lightchain of serotype A based on wild-type Clostridium botulinum sequence 47Met Val Gln Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn 1 5 1015 Gly Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln 20 2530 Pro Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu 35 4045 Arg Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro 50 5560 Glu Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser 65 7075 80 Thr Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe 8590 95 Glu Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile100 105 110 Val Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr GluLeu 115 120 125 Lys Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro AspGly Ser 130 135 140 Tyr Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly ProSer Ala Asp 145 150 155 160 Ile Ile Gln Phe Glu Cys Lys Ser Phe Gly HisGlu Val Leu Asn Leu 165 170 175 Thr Arg Asn Gly Tyr Gly Ser Thr Gln TyrIle Arg Phe Ser Pro Asp 180 185 190 Phe Thr Phe Gly Phe Glu Glu Ser LeuGlu Val Asp Thr Asn Pro Leu 195 200 205 Leu Gly Ala Gly Lys Phe Ala ThrAsp Pro Ala Val Thr Leu Ala His 210 215 220 Glu Leu Ile His Ala Gly HisArg Leu Tyr Gly Ile Ala Ile Asn Pro 225 230 235 240 Asn Arg Val Phe LysVal Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly 245 250 255 Leu Glu Val SerPhe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala 260 265 270 Lys Phe IleAsp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr 275 280 285 Asn LysPhe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile 290 295 300 ValGly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu 305 310 315320 Lys Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys 325330 335 Leu Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu340 345 350 Asp Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr TyrLeu 355 360 365 Asn Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro LysVal Asn 370 375 380 Tyr Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr AsnLeu Ala Ala 385 390 395 400 Asn Phe Asn Gly Gln Asn Thr Glu Ile Asn AsnMet Asn Phe Thr Lys 405 410 415 Leu Lys Asn Phe Thr Gly Leu Phe Glu PheTyr Lys Leu Leu Cys Val 420 425 430 Arg Gly Ile Ile Thr Ser Lys Thr LysSer Leu Asp Lys Gly Tyr Asn 435 440 445 Lys

We claim:
 1. A method for producing a botulinum neurotoxin light chaincomprising: culturing a host cell comprising a DNA molecule encoding thebotulinum neurotoxin light chain, the DNA molecule having a nucleotidesequence expressible in the host cell, at a temperature below 30° C.,wherein the DNA molecule is expressed and the light chain is produced,and isolating the botulinum neurotoxin light chain.
 2. A method forproducing a botulinum neurotoxin light chain comprising: culturing ahost cell comprising a DNA molecule encoding the botulinum neurotoxinlight chain, the DNA molecule having a nucleic acid sequence expressiblein the host cell, at about 18° C., wherein DNA molecule is expressed andthe botulinum neurotoxin light chain is produced, and isolating thebotulinum neurotoxin light chain.
 3. The method of claim 1 or 2 whereinthe host cell is selected from the group consisting of Escherichia coliand Pichia pastoris.
 4. The method of claim 1 or 2 wherein the host cellis Escherichia coli.
 5. The method of claim 1 or 2 further comprisingobtaining an insoluble protein fraction from the cultured host cell andpurifying the botulinum neurotoxin light chain from the insolubleprotein fraction.
 6. The method of claim 5 further comprisingsolubilizing the insoluble protein fraction.
 7. The method of claim 6further comprising subjecting the solubilized insoluble protein fractionto cation exchange chromatography under conditions such that a purifiedpreparation of botulinum neurotoxin light chain is obtained.
 8. Themethod of claim 1 or 2 further comprising obtaining a soluble proteinfraction from the cultured host cell and purifying the botulinumneurotoxin light chain from the soluble protein fraction.
 9. The methodof claim 8 further comprising subjecting the soluble protein fraction tocation exchange chromatography under conditions such that a purifiedpreparation of botulinum neurotoxin light chain is obtained.
 10. Themethod of claim 5 or 8 wherein more than about 100 mg of purifiedbotulinum neurotoxin light chain is obtained per liter of culture. 11.The method of claim 10 wherein more than 500 mg of purified botulinumneurotoxin light chain is obtained per liter of culture.
 12. The methodof claim 11 wherein about 1 gram of purified botulinum neurotoxin lightchain is obtained per liter of culture.
 13. The method of claim 5 or 8wherein the purified botulinum neurotoxin light chain is catalyticallyactive.
 14. The method of claim 1 or 2 wherein the DNA Molecules has thenucleic acid sequence of bases 9-1337 of SEQ ID NO:4.
 15. The method ofclaim 1 or 2 wherein the DNA molecules encodes a botulinum neurotoxinlight chain perotype A.
 16. The method of claim 1 or 2 wherein the DNAmolecule encodes a botulinum neurotoxin light chain selected from thegroup consisting of botulinum neurotoxin light chain serotype B,botulinum neurotoxin light chain serotype C₁, botulinum neurotoxin lightchain serotype D, botulinum neurotoxin light chain serotype E, botulinumneurotoxin light chain serotype F, and botulinum neurotoxin light chainserotype G.
 17. The method of claim 15 wherein the nucleic acid has atotal A+T content that is less than about 70% 18.The method of claim 17wherein the nucleic acid is selected from the group consisting of SEQ IDNO:47 and SEQ ID NO:
 21. 19. The method of claim 17 wherein the A+Tcontent of any 50 consecutive nucleotides of the nucleic acid moleculeis less than about 75%.
 20. The method of claim 18 wherein the A+Tcontent of any 50 consecutive nucleotides of the nucleic acid moleculeis less than about 75%.
 21. The method of claim 16 wherein the nucleicacid molecule has a a nucleic acid sequence selected from the groupconsisting of SEQ ID NOS:6, 8, 10, 12, 14, 16, 22, 26, 30, 34, 38, and42.
 22. The method of claim 16 wherein the nucleic acid has a total A+Tcontent that is less than about 70%.
 23. The method of claim 22 whereinthe A+T content of any 50 consecutive nucleotides of the nucleic acidmolecule is less than about 75%.
 24. The method of claim 22 wherein thenucleic acid molecule encodes a polypeptide having an amino acidsequence selected from the group consisting of SEQ ID NO:7, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:23,SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:35, SEQ ID NO:39, and SEQ IDNO:43, wherein the nucleic acid has a total A+T content that is lessthan about 70%.